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Viability of the sporocysts of Sarcocystis cruzi after exposure to different temperatures and relative humidities
veterinary parasitology ELSEVIER Veterinary Parasitology 67 (1996) 153-160
Viability of the sporocysts of Sarcocystis cruzi after exposure to different temperatures and relative humidities G. Savini a,*, I.D. Robertson b, J.D. Dunsmore b a lstituto Zooprofilattico Sperimentale delrAbruzzo e Molise, Teramo, Italy b School o f Veterinary Studies, Murdoch University, Murdoch, W.A. 6150, Australia
Received 11 September 1995; accepted 8 May 1996
Abstract The effect of temperature and relative humidity (RH) on the survival of sporocysts of S. cruzi were investigated in vitro. Under all experimental conditions (temperature of 4°C, 37°C, or room temperature; RH of 18%, 75%, or 100%) some sporozoites retained their viability to excyst for at least 90 days. The best conditions for survival were 4°C at 100% RH (more than 240 days) and 37°C at 18% RH (more than 180 days). Sporocysts maintained at room temperature at all humidities had the lowest level of survival. It is concluded that sporocysts of S. cruzi are able to survive in most environments for several months and that the fluctuation of the daily ambient temperature is likely to influence the viability of the sporocysts. Keywords: Sarcocystis cruzi; Sporocysts
I. Introduction
Sarcocystis spp. are intracellular protozoan organisms infecting animals and humans throughout the world (Dubey et al., 1989). In order to perpetuate the life cycle, Sarcocystis must be transmitted from an omnivorous or herbivorous intermediate host to a carnivorous definitive host and back again. This is achieved by sarcocysts in the former and sporocysts in the latter (Levine, 1988). Survival of stages in the environment * Corresponding author. Tel: 0039 861 332248; fax: 0039 861 332251. 0304-4017/96//$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S0304-401 7(96)01 046- I
154
G. Savini et al./Veterinary Parasitology 67 (1996) 153-160
is therefore likely to be a crucial factor in the epidemiology of sarcocystiosis. As sporocysts of Sarcocystis are sporulated and small, they are resistant to many environmental factors and their ability to disperse in the environment is of significant epidemiological importance. Attempts to control the prevalence of sarcocystiosis in farm animals must consider the ability of sporocysts to be affected by various external factors. For coccidial oocysts, heat and drying are deleterious and their period of survival is a function of the period of exposure to these factors (Fayer, t980). However, for sporocysts of Sarcocystis only meagre data about survival at various temperatures and relative humidities are available (Bergler et al., 1980; Heydorn, 1980; McKenna and Charleston, 1992). In previous work it has been emphasised how climate affects the distribution of infection with S. cruzi in Western Australian cattle (Savini et al., 1992). As sporocysts play an essential role in perpetuating the infection, it is important to know more about the ability of this stage to survive under various environmental conditions. Determining the viability of sporocysts is not easy since they are not motile. Several methods have been used to determine the viability of sporocysts which have included excystation in vitro, differential staining of live and dead material and the use of artificial infection of animals. While each of these methods has its own advantages, none is rapid, simple, and reliable. The ability to induce infection in a susceptible host is the essential test of the viability of any infectious agent, however the cost, time and large numbers of sporocysts required to perform animal infectivity studies, along with the difficulties in maintaining intermediate hosts free of extraneous infections during the long development period of the infection with Sarcocystis, makes the method impractical for routine use. The capability of sporocysts to take up stain such as methylene blue and trypan blue has also been used. However, their inaccuracy in the identification of live and dead organisms and the very short exposure time (1 min) after which excysted sporozoites could no longer be recognised, have limited their usefulness (Bergler et al., 1980). Because sporozoites must be released from the sporocysts to infect the definitive host, the ability to excyst and the proportion that excyst have been used for many years (Goodrich, 1944) as an indicator of their ability to survive. However, the reliability of this method was questioned by Heydorn (1980) who revealed that viability in vitro was not wholly reflected by infectivity in vivo. He concluded that not all of the excysted sporozoites were infective to the intermediate host. Although concurring with this finding, McKenna and Charleston (1992) believed that the ability to excyst was still a satisfactory criterion for assessing the viability of the sporocysts of Sarcocystis spp. The possibility of considering, incorrectly, a sporocyst infective when it was not, was considered preferable to considering a sporocyst non-infective when it actually was infective. In other words, it is better to overestimate than underestimate the risk of infection from the sporocysts. The objective of this study was to test the rate of survival of sporocysts of S. cruzi after exposure to various temperatures and relative humidities. In the absence of other more practical methods, the ability to excyst in vitro was the method chosen for measuring survival. A fluorescent dye exclusion method using fluorescein diacetate (FDA)-propidium iodide (PI) was alternatively tested as a more practical and reliable tool to assess the viability of Sarcocystis sporocysts.
G. Savini et aL / Veterinary Parasitology 67 (1996) 153-160
155
2. Materials and methods
Sporocysts of S. cruzi were recovered from dog faeces as described by Dubey et al. (1989) and stored at 4°C in HBSS with antibiotic and fungizone for 10 months until use. 2.1. Excystation Excystation was achieved using the method which resulted in the best excystation rate for S. cruzi as described by Savini et al. (1994). In brief, sporocysts were pretreated with 2.6% sodium hypochlorite (NaOC1) for 30 min and then incubated for 4-8 h in an excysting fluid containing 2% trypsin, 5% sodium taurocholate in HBSS to which 0.3 M sodium bicarbonate in distilled water was mixed. The excystation rate (ER) (percentage of excysted sporozoites) was determined by dividing the number of free sporozoites by the sum of the number of sporozoites in sporocysts and free sporozoites and recording it as a percentage. 2.2. Dye test The staining solutions were made following the method of Jones and Sneft (1985). A stock solution of FDA was made by mixing 10 mg of FDA with 1 ml of acetone. A working solution was prepared by adding 0.04 ml of the FDA stock solution to 10 ml Dulbecco phosphate-buffered saline at a pH of 7. A stock solution of P! was made by mixing 0.5 mg of PI with 50 ml of Dulbecco phosphate-buffered saline at a pH of 7. Pellets of sporocysts of S. cruzi were stained with these dyes at concentrations of either 4, 8, 12, or 40 /xg of FDA per 10 6 sporocysts or 3, 6, 9 or 30 /xg of PI per 10 6 sporocysts. Sporocysts were stained for 5 min before microscopic observation. Stained slides were examined with a fluorescence microscope at excitation wavelengths of 455 to 490 nm for FDA and 545 to 546 nm for P1 to detect viable (yellow-green) and non-viable (red) sporocysts in a two colour epifluorescence. Fluorogenic dyes have been accepted as a sensitive means for the determination of cyst viability in many protozoan parasites (Jackson et al., 1985; Schupp and Erlandsen, 1987). 2.3. Relative humidity Two different relative humidities maintained at three different temperatures were established using sealed containers containing saturated solutions (Winston and Bates, 1960). Sludges of sodium and lithium chloride were chosen for their ability to produce fairly constant relative humidities at different temperatures. Expected levels of humidity of 75% were obtained in the containers with saturated solutions of sodium chloride whereas slightly higher than expected relative humidity, within the range of 18-20% rather than 11-15%, were found in the containers with lithium chloride. At constant intervals both before and during the experiment, the humidity levels within the container were checked by using a humidity indicator (HMl 11; Vaisala; Paton, Sydney, SA). No significant deviations (in the range of 0.1-0.2%) from the initial humidity were recorded during the experiment.
G. Savini et al. / Veterinary Parasitology 67 (1996) 153-160
156
2.4. Experimental procedure Fifty thousand 10-month-old sporocysts, suspended in 100 /xl of tap water, were placed onto the concavity of each of 216 cavity slides and dried out with an electric fan. After the surface water evaporated, these slides were randomly divided into nine groups of 24 slides each. One group was placed into each of six sealed containers. The remaining 72 slides were divided into nine groups and each was washed with tap water. The washing from each of the slides in the nine groups was pooled together by group into a 10 ml centrifuge tube and centrifuged at 2000 RPM for 10 min. The pellets then were resuspended in 8 ml of tap water and were maintained at the same temperature as the containers and used as undesiccated controls (100% RH). One container of slides for each saturated solution and three control centrifuge tubes were then placed in an incubator at 37°C, in a refrigerator at 4°C and maintained at room temperature. The experiment was performed during the summer months of November-March when the room temperature ranged from 18 to 34°C. Seven days after the beginning of the experiment and then at 30 day intervals three slides from each container and 1 ml from each control tube were removed. The slides were subsequently rehydrated and the material of each slide collected into a 10 ml centrifuge tube. The tubes were centrifuged and the pellets washed before exposure to the excysting procedures.
3. Results Under all experimental conditions (temperature 4°C or 37°C or room temperature; RH 18% or 75% or 100%) some sporocysts retained their viability to excyst for at least 90 days (Figs. 1-6). The loss of viability was more marked at room temperature than at either of the constant temperatures tested.
1 O0 9o 18% RH
~
80
75% RH
70
I~%RH
~
80
~ _a. m
50
~
40 ~o
20 10 0 0
30
75
120
165
210
255
Days Fig. 1. E f f e c t o f d e s i c c a t i o n on the e x c y s t a t i o n o f s p o r o c y s t s o f S. cruzi m a i n t a i n e d at 4 ° C a n d v a r y i n g r e l a t i v e humidities.
G. Savini et al. / Veterinary Parasitology 67 (1996) 153-160
157
1.00 90 .80 •
18°/*RH 75%RH
+
.70
100% RH
60 50 .40 30 20 .10 0
0
30
75
120
165
210
255
Days
Fig. 2. Effect of desiccation on the excystation of sporocysts o f S. cruzi maintained at 37°C and varying
relative humidities. 1.00 .90
.80 70
.60
"0"-
18%RH 75%RH
"-Ii--
100% RH
--~ I .50 ~
.40
~1
30 .20 .10 0
30
0
75
120
, w
• . . . .
165
~
210
•
255
.
Days
F i g . 3. E f f e c t o f d e s i c c a t i o n o n the e x c y s t a t i o n o f s p o r o c y s t s o f S. cruzi m a i n t a i n e d at r o o m t e m p e r a t u r e a n d varying relative humidities.
90 1.80 .00
~
7
"
C
70 .60 50 ~
\
~
\
+
roomtempersture
.30 .20 .10 0
.
0
.
.
.
.
30
.
.
.
.
75
I " ~ .
120
&
.
165
A
.
.
210
.
, . .
.
255
Days
Fig. 4. Effect of temperature on the excystation of sporocysts of S. cruzi maintained at 100% relative humidity.
G. Sauini et al. / Veterinary Parasitology 67 (1996) 153-160
158 1 oo 9o
Jlp
~
370C
. , o ~
o
~
60
40 ~o
20 10 0
30
75
120
165
210
255
Days
Fig. 5. Effect o f temperature on the excystation of sporocysts o f S. cruzi maintained at 75% relative humidity.
The best condition for survival was 4°C at 100% RH (Fig. 1 and Fig. 4). Under these conditions there was no decline in excystation to day 240. Conversely, at 100% RH survival had declined to zero by 120 days at 37°C and at room temperature (Fig. 4). Perhaps the most interesting results were those obtained at the low RH of 18% (Fig. 6) where survival was good up to 180 days at both 4°C and 37°C. Low temperatures were also compatible with survival and at 4°C there was no decline in viability by 150 days. Beyond this point there was a decline in excystability except in a saturated atmosphere (Fig. 1). Cysts maintained at room temperature had the lowest level of survival and at all humidities survival was low even at Day 60 (Fig. 3). In contrast at 37°C at all humidities there was reasonable survival to Day 60 (Fig. 2), Neither sporocysts nor excysted sporozoites absorbed the stain when stained at any concentration of dye.
1 oo 90
80-
O
~ -
37~
~
room tem~rature
,--B--
4~
7060
"~
50
"
40 30
-
20
~
10
0
30
75
120
165
210
255
Days
Fig. 6. E f f e c t o f temperature on the excystation of sporocysts o f S. cruzi maintained at 18% relative humidity.
G. Saoini et al./Veterinary Parasitology 67 (1996) 153-160
159
4. Discussion
Grazing animals can only become infected with Sarcocystis after ingestion of oocysts or sporocysts which have been shed in the faeces of the appropriate infected definitive host. Clearly the level of infection in areas grazed by the intermediate host is dependent on the number of sporocysts shed into the area and their survivability. Survivability is largely climate-dependent (Fayer, 1980). In areas which are not densely populated by the two host species it is likely that transmission occurs mainly at sites where species congregate, possibly only once or twice a year such as stock yards. Hence the epidemiology of the infection will be greatly affected by the capacity of the sporocysts to survive for long periods under possibly harsh environmental conditions. In all situations the sporocysts must rely on their ability to survive in order to ensure the completion of the life cycle. As a consequence of the results of the epidemiological study undertaken on Western Australian cattle (Savini et al., 1992) and other studies (Bergler et al., 1980; Heydorn, 1980) on the viability of the Sarcocystis sporocysts, it could be expected that dryness and high temperatures have a deleterious effect on sporocysts of S. cruzi. In many studies, the ability of sporocysts to survive desiccation was frequently measured in days and was directly proportional to the relative humidity to which they were exposed (Bergler et al., 1980; Heydorn, 1980). However, in the present study, sporocysts of S. cruzi were shown to be capable of surviving desiccation for months, with the length of survival being related to both temperature and relative humidity. The finding that sporocysts were able to survive for longer periods at low humidity, particularly at a high temperature, is not unique. Similar results have been observed in sporocysts of S. gigantea (McKenna and Charleston, 1992) and with the eggs of the plant nematode Globodera (Heterodera) rostochiensis and infective larvae of Trichostrongylus colubriformis (Wharton, 1982) and Nematodirus battus (Parkin, 1976). For the eggs of the plant nematode it has been suggested that low relative humidity may reduce the permeability of the external wall of the resting stages of these parasites which, in turn, may lower the loss of water (Ellenby, 1968). McKenna and Charleston (1992) speculated that the wall of the sporocyst of S. gigantea may possess properties which aid in the survival of their sporozoites at times of severe desiccation as well as increasing their tolerance to environmental temperatures. According to the results of this study it appears that sporocysts of S. cruzi may also have these properties. However, the possibility that at higher temperatures and relative humidities the sporocysts may have died because of lack of oxygen cannot be discarded. At these temperatures and relative humidities the number of aerobic bacteria which compete with sporozoites for respiratory demands increase (McKenna and Charleston, 1992). The results of this study, which show that sporocysts of S. cruzi are able to remain viable for long periods after being exposed to high temperatures and low relative humidities, appear to be inconsistent with the distribution of infection in Western Australian cattle. However, the finding that fluctuations in temperatures may have a deleterious effect on the viability of sporocysts does match field data. The areas where the prevalence of Sarcocystis infection has been found to be low were those characterised by great fluctuations between minimum and maximum daily temperatures.
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Fluorogenic dyes have been accepted as a sensitive means for determining the viability of cysts of many protozoan parasites (Jackson et al., 1985: Schupp and Erlandsen, 1987). The results obtained in this study were disappointing because the fluorogenic staining procedure would have been rapid, simple and inexpensive. In addition it would have allowed testing the effects of disinfectants and various environmental factors on the viability of the cysts both before and after exposure to chemical and physical agents. It has been successfully employed for other protozoans such as Giardia, Trypanosoma and Leishmania (Jackson et al., 1985). This result may be associated with the different structure of the sporocyst wall of Sarcocystis which may prevent the fluorescein from entering the sporocyst. Although some factors such as the direct action of sunlight, and the competition by other faecal parasites or microflora (Fayer, 1980), have a deleterious effect on the survival of sporocysts in the natural environment, the results of the present study suggest that sporocysts of S. cruzi are able to remain infective for potential intermediate hosts for several months. This period of infectivity is likely to be longer in cooler climates but may be reduced when temperatures greatly fluctuate.
References Bergler, K.G., Erber, H. and Boch, J., 1980. Untersuchungen zur /t)berlebensfiihigkeit von Sporozysten bzw. Oozysten yon Sarcocystis, Toxoplasma, Hammondia und Eimeria unter Laborund Freilandbedingungen. Berl. Mi~nch Tier'~irztl Wochenschr., 93: 288-293. Dubey, J.P., Speer, C.A. and Fayer, R., 1989. Sarcocystosis of Animals and Man. CRC Press, Boca Raton, FL. Ellenby, C., 1968. Desiccation survival in the plant parasitic nematodes, Heterodera rostochiensis Wollenweber and Ditylenchus dipsaci (Kuhn) Filipjev. Proc. R. Soc. Lond. B, 169: 203-213. Fayer, R., 1980. Epidemiology of protozoan infections: the coccidia. Vet. Parasitol., 6: 75-103. Goodrich, H.P., 1944. Coccidian oocysts. Parasitology, 36: 72-79. Heydom, A.O., 1980. Zur Widerstandsfahigkeit von Sarcocystis bovicanis-Sporozysten. Berl. Miinch. Tief~rztl. Wochenschr., 93: 267-270. Jackson, P.R., Pappas, M.G. and Hansen, B.D., 1985. Fluorogenic subs(rate detection of viable intracellular and extracellular pathogenic protozoa. Science, 227: 435-438. Jones, K.H. and Sneft, J.A., 1985. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J. Histochem. Cytochem., 33:77 79. Levine, N.D., 1988. The Protozoan Phylum Apicomplexa. CRC Press, Boca Raton, FL. McKenna, P.B. and Charleston, W.A.G., 1992. The survival of Sarcocystis gigantea sporocysts following exposure to various chemical and physical agents. Vet. Parasitol., 45: 1-16. Parkin, J.T., 1976. The effect of moisture supply upon the development and hatching of the eggs of Nematodirus battus. Parasitology, 73: 353-354. Savini, G., Dunsmore, J.D., Robertson, I.D. and Seneviratna P., 1992. Epidemiology of Sarco(3~stis spp. in cattle of Western Australia. Epidemiol. Infect., 108:107-113. Savini, G., Dunsmore, .I.D. and Robertson, I.D., 1994. Evaluation of a serological test system for the diagnosis of Sarcocystis cruzi infection in cattle using S. cruzi merozoite antigen. Vet. Parasitol., 51:181 189. Schupp, D.G. and Erlandsen, S.L., 1987. A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity. Appl. Envir. Microbiol., 53: 704-707. Wharton, D.A., 1982. The survival of desiccation by the free-living stages of Trichostrongylus colubriJormis (Nematoda: Trichostrongilidae). Parasitology, 84: 455-462. Winston, P.W. and Bates, D., 1960. Saturated solutions for control of humidity in biological research. Ecology, 41: 232-237.
Viability of the sporocysts of Sarcocystis cruzi after exposure to different temperatures and relative humidities G. Savini a,*, I.D. Robertson b, J.D. Dunsmore b a lstituto Zooprofilattico Sperimentale delrAbruzzo e Molise, Teramo, Italy b School o f Veterinary Studies, Murdoch University, Murdoch, W.A. 6150, Australia
Received 11 September 1995; accepted 8 May 1996
Abstract The effect of temperature and relative humidity (RH) on the survival of sporocysts of S. cruzi were investigated in vitro. Under all experimental conditions (temperature of 4°C, 37°C, or room temperature; RH of 18%, 75%, or 100%) some sporozoites retained their viability to excyst for at least 90 days. The best conditions for survival were 4°C at 100% RH (more than 240 days) and 37°C at 18% RH (more than 180 days). Sporocysts maintained at room temperature at all humidities had the lowest level of survival. It is concluded that sporocysts of S. cruzi are able to survive in most environments for several months and that the fluctuation of the daily ambient temperature is likely to influence the viability of the sporocysts. Keywords: Sarcocystis cruzi; Sporocysts
I. Introduction
Sarcocystis spp. are intracellular protozoan organisms infecting animals and humans throughout the world (Dubey et al., 1989). In order to perpetuate the life cycle, Sarcocystis must be transmitted from an omnivorous or herbivorous intermediate host to a carnivorous definitive host and back again. This is achieved by sarcocysts in the former and sporocysts in the latter (Levine, 1988). Survival of stages in the environment * Corresponding author. Tel: 0039 861 332248; fax: 0039 861 332251. 0304-4017/96//$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S0304-401 7(96)01 046- I
154
G. Savini et al./Veterinary Parasitology 67 (1996) 153-160
is therefore likely to be a crucial factor in the epidemiology of sarcocystiosis. As sporocysts of Sarcocystis are sporulated and small, they are resistant to many environmental factors and their ability to disperse in the environment is of significant epidemiological importance. Attempts to control the prevalence of sarcocystiosis in farm animals must consider the ability of sporocysts to be affected by various external factors. For coccidial oocysts, heat and drying are deleterious and their period of survival is a function of the period of exposure to these factors (Fayer, t980). However, for sporocysts of Sarcocystis only meagre data about survival at various temperatures and relative humidities are available (Bergler et al., 1980; Heydorn, 1980; McKenna and Charleston, 1992). In previous work it has been emphasised how climate affects the distribution of infection with S. cruzi in Western Australian cattle (Savini et al., 1992). As sporocysts play an essential role in perpetuating the infection, it is important to know more about the ability of this stage to survive under various environmental conditions. Determining the viability of sporocysts is not easy since they are not motile. Several methods have been used to determine the viability of sporocysts which have included excystation in vitro, differential staining of live and dead material and the use of artificial infection of animals. While each of these methods has its own advantages, none is rapid, simple, and reliable. The ability to induce infection in a susceptible host is the essential test of the viability of any infectious agent, however the cost, time and large numbers of sporocysts required to perform animal infectivity studies, along with the difficulties in maintaining intermediate hosts free of extraneous infections during the long development period of the infection with Sarcocystis, makes the method impractical for routine use. The capability of sporocysts to take up stain such as methylene blue and trypan blue has also been used. However, their inaccuracy in the identification of live and dead organisms and the very short exposure time (1 min) after which excysted sporozoites could no longer be recognised, have limited their usefulness (Bergler et al., 1980). Because sporozoites must be released from the sporocysts to infect the definitive host, the ability to excyst and the proportion that excyst have been used for many years (Goodrich, 1944) as an indicator of their ability to survive. However, the reliability of this method was questioned by Heydorn (1980) who revealed that viability in vitro was not wholly reflected by infectivity in vivo. He concluded that not all of the excysted sporozoites were infective to the intermediate host. Although concurring with this finding, McKenna and Charleston (1992) believed that the ability to excyst was still a satisfactory criterion for assessing the viability of the sporocysts of Sarcocystis spp. The possibility of considering, incorrectly, a sporocyst infective when it was not, was considered preferable to considering a sporocyst non-infective when it actually was infective. In other words, it is better to overestimate than underestimate the risk of infection from the sporocysts. The objective of this study was to test the rate of survival of sporocysts of S. cruzi after exposure to various temperatures and relative humidities. In the absence of other more practical methods, the ability to excyst in vitro was the method chosen for measuring survival. A fluorescent dye exclusion method using fluorescein diacetate (FDA)-propidium iodide (PI) was alternatively tested as a more practical and reliable tool to assess the viability of Sarcocystis sporocysts.
G. Savini et aL / Veterinary Parasitology 67 (1996) 153-160
155
2. Materials and methods
Sporocysts of S. cruzi were recovered from dog faeces as described by Dubey et al. (1989) and stored at 4°C in HBSS with antibiotic and fungizone for 10 months until use. 2.1. Excystation Excystation was achieved using the method which resulted in the best excystation rate for S. cruzi as described by Savini et al. (1994). In brief, sporocysts were pretreated with 2.6% sodium hypochlorite (NaOC1) for 30 min and then incubated for 4-8 h in an excysting fluid containing 2% trypsin, 5% sodium taurocholate in HBSS to which 0.3 M sodium bicarbonate in distilled water was mixed. The excystation rate (ER) (percentage of excysted sporozoites) was determined by dividing the number of free sporozoites by the sum of the number of sporozoites in sporocysts and free sporozoites and recording it as a percentage. 2.2. Dye test The staining solutions were made following the method of Jones and Sneft (1985). A stock solution of FDA was made by mixing 10 mg of FDA with 1 ml of acetone. A working solution was prepared by adding 0.04 ml of the FDA stock solution to 10 ml Dulbecco phosphate-buffered saline at a pH of 7. A stock solution of P! was made by mixing 0.5 mg of PI with 50 ml of Dulbecco phosphate-buffered saline at a pH of 7. Pellets of sporocysts of S. cruzi were stained with these dyes at concentrations of either 4, 8, 12, or 40 /xg of FDA per 10 6 sporocysts or 3, 6, 9 or 30 /xg of PI per 10 6 sporocysts. Sporocysts were stained for 5 min before microscopic observation. Stained slides were examined with a fluorescence microscope at excitation wavelengths of 455 to 490 nm for FDA and 545 to 546 nm for P1 to detect viable (yellow-green) and non-viable (red) sporocysts in a two colour epifluorescence. Fluorogenic dyes have been accepted as a sensitive means for the determination of cyst viability in many protozoan parasites (Jackson et al., 1985; Schupp and Erlandsen, 1987). 2.3. Relative humidity Two different relative humidities maintained at three different temperatures were established using sealed containers containing saturated solutions (Winston and Bates, 1960). Sludges of sodium and lithium chloride were chosen for their ability to produce fairly constant relative humidities at different temperatures. Expected levels of humidity of 75% were obtained in the containers with saturated solutions of sodium chloride whereas slightly higher than expected relative humidity, within the range of 18-20% rather than 11-15%, were found in the containers with lithium chloride. At constant intervals both before and during the experiment, the humidity levels within the container were checked by using a humidity indicator (HMl 11; Vaisala; Paton, Sydney, SA). No significant deviations (in the range of 0.1-0.2%) from the initial humidity were recorded during the experiment.
G. Savini et al. / Veterinary Parasitology 67 (1996) 153-160
156
2.4. Experimental procedure Fifty thousand 10-month-old sporocysts, suspended in 100 /xl of tap water, were placed onto the concavity of each of 216 cavity slides and dried out with an electric fan. After the surface water evaporated, these slides were randomly divided into nine groups of 24 slides each. One group was placed into each of six sealed containers. The remaining 72 slides were divided into nine groups and each was washed with tap water. The washing from each of the slides in the nine groups was pooled together by group into a 10 ml centrifuge tube and centrifuged at 2000 RPM for 10 min. The pellets then were resuspended in 8 ml of tap water and were maintained at the same temperature as the containers and used as undesiccated controls (100% RH). One container of slides for each saturated solution and three control centrifuge tubes were then placed in an incubator at 37°C, in a refrigerator at 4°C and maintained at room temperature. The experiment was performed during the summer months of November-March when the room temperature ranged from 18 to 34°C. Seven days after the beginning of the experiment and then at 30 day intervals three slides from each container and 1 ml from each control tube were removed. The slides were subsequently rehydrated and the material of each slide collected into a 10 ml centrifuge tube. The tubes were centrifuged and the pellets washed before exposure to the excysting procedures.
3. Results Under all experimental conditions (temperature 4°C or 37°C or room temperature; RH 18% or 75% or 100%) some sporocysts retained their viability to excyst for at least 90 days (Figs. 1-6). The loss of viability was more marked at room temperature than at either of the constant temperatures tested.
1 O0 9o 18% RH
~
80
75% RH
70
I~%RH
~
80
~ _a. m
50
~
40 ~o
20 10 0 0
30
75
120
165
210
255
Days Fig. 1. E f f e c t o f d e s i c c a t i o n on the e x c y s t a t i o n o f s p o r o c y s t s o f S. cruzi m a i n t a i n e d at 4 ° C a n d v a r y i n g r e l a t i v e humidities.
G. Savini et al. / Veterinary Parasitology 67 (1996) 153-160
157
1.00 90 .80 •
18°/*RH 75%RH
+
.70
100% RH
60 50 .40 30 20 .10 0
0
30
75
120
165
210
255
Days
Fig. 2. Effect of desiccation on the excystation of sporocysts o f S. cruzi maintained at 37°C and varying
relative humidities. 1.00 .90
.80 70
.60
"0"-
18%RH 75%RH
"-Ii--
100% RH
--~ I .50 ~
.40
~1
30 .20 .10 0
30
0
75
120
, w
• . . . .
165
~
210
•
255
.
Days
F i g . 3. E f f e c t o f d e s i c c a t i o n o n the e x c y s t a t i o n o f s p o r o c y s t s o f S. cruzi m a i n t a i n e d at r o o m t e m p e r a t u r e a n d varying relative humidities.
90 1.80 .00
~
7
"
C
70 .60 50 ~
\
~
\
+
roomtempersture
.30 .20 .10 0
.
0
.
.
.
.
30
.
.
.
.
75
I " ~ .
120
&
.
165
A
.
.
210
.
, . .
.
255
Days
Fig. 4. Effect of temperature on the excystation of sporocysts of S. cruzi maintained at 100% relative humidity.
G. Sauini et al. / Veterinary Parasitology 67 (1996) 153-160
158 1 oo 9o
Jlp
~
370C
. , o ~
o
~
60
40 ~o
20 10 0
30
75
120
165
210
255
Days
Fig. 5. Effect o f temperature on the excystation of sporocysts o f S. cruzi maintained at 75% relative humidity.
The best condition for survival was 4°C at 100% RH (Fig. 1 and Fig. 4). Under these conditions there was no decline in excystation to day 240. Conversely, at 100% RH survival had declined to zero by 120 days at 37°C and at room temperature (Fig. 4). Perhaps the most interesting results were those obtained at the low RH of 18% (Fig. 6) where survival was good up to 180 days at both 4°C and 37°C. Low temperatures were also compatible with survival and at 4°C there was no decline in viability by 150 days. Beyond this point there was a decline in excystability except in a saturated atmosphere (Fig. 1). Cysts maintained at room temperature had the lowest level of survival and at all humidities survival was low even at Day 60 (Fig. 3). In contrast at 37°C at all humidities there was reasonable survival to Day 60 (Fig. 2), Neither sporocysts nor excysted sporozoites absorbed the stain when stained at any concentration of dye.
1 oo 90
80-
O
~ -
37~
~
room tem~rature
,--B--
4~
7060
"~
50
"
40 30
-
20
~
10
0
30
75
120
165
210
255
Days
Fig. 6. E f f e c t o f temperature on the excystation of sporocysts o f S. cruzi maintained at 18% relative humidity.
G. Saoini et al./Veterinary Parasitology 67 (1996) 153-160
159
4. Discussion
Grazing animals can only become infected with Sarcocystis after ingestion of oocysts or sporocysts which have been shed in the faeces of the appropriate infected definitive host. Clearly the level of infection in areas grazed by the intermediate host is dependent on the number of sporocysts shed into the area and their survivability. Survivability is largely climate-dependent (Fayer, 1980). In areas which are not densely populated by the two host species it is likely that transmission occurs mainly at sites where species congregate, possibly only once or twice a year such as stock yards. Hence the epidemiology of the infection will be greatly affected by the capacity of the sporocysts to survive for long periods under possibly harsh environmental conditions. In all situations the sporocysts must rely on their ability to survive in order to ensure the completion of the life cycle. As a consequence of the results of the epidemiological study undertaken on Western Australian cattle (Savini et al., 1992) and other studies (Bergler et al., 1980; Heydorn, 1980) on the viability of the Sarcocystis sporocysts, it could be expected that dryness and high temperatures have a deleterious effect on sporocysts of S. cruzi. In many studies, the ability of sporocysts to survive desiccation was frequently measured in days and was directly proportional to the relative humidity to which they were exposed (Bergler et al., 1980; Heydorn, 1980). However, in the present study, sporocysts of S. cruzi were shown to be capable of surviving desiccation for months, with the length of survival being related to both temperature and relative humidity. The finding that sporocysts were able to survive for longer periods at low humidity, particularly at a high temperature, is not unique. Similar results have been observed in sporocysts of S. gigantea (McKenna and Charleston, 1992) and with the eggs of the plant nematode Globodera (Heterodera) rostochiensis and infective larvae of Trichostrongylus colubriformis (Wharton, 1982) and Nematodirus battus (Parkin, 1976). For the eggs of the plant nematode it has been suggested that low relative humidity may reduce the permeability of the external wall of the resting stages of these parasites which, in turn, may lower the loss of water (Ellenby, 1968). McKenna and Charleston (1992) speculated that the wall of the sporocyst of S. gigantea may possess properties which aid in the survival of their sporozoites at times of severe desiccation as well as increasing their tolerance to environmental temperatures. According to the results of this study it appears that sporocysts of S. cruzi may also have these properties. However, the possibility that at higher temperatures and relative humidities the sporocysts may have died because of lack of oxygen cannot be discarded. At these temperatures and relative humidities the number of aerobic bacteria which compete with sporozoites for respiratory demands increase (McKenna and Charleston, 1992). The results of this study, which show that sporocysts of S. cruzi are able to remain viable for long periods after being exposed to high temperatures and low relative humidities, appear to be inconsistent with the distribution of infection in Western Australian cattle. However, the finding that fluctuations in temperatures may have a deleterious effect on the viability of sporocysts does match field data. The areas where the prevalence of Sarcocystis infection has been found to be low were those characterised by great fluctuations between minimum and maximum daily temperatures.
160
G. Savtni et a[. / Veterinary Parasitology 67 (1996) 153-160
Fluorogenic dyes have been accepted as a sensitive means for determining the viability of cysts of many protozoan parasites (Jackson et al., 1985: Schupp and Erlandsen, 1987). The results obtained in this study were disappointing because the fluorogenic staining procedure would have been rapid, simple and inexpensive. In addition it would have allowed testing the effects of disinfectants and various environmental factors on the viability of the cysts both before and after exposure to chemical and physical agents. It has been successfully employed for other protozoans such as Giardia, Trypanosoma and Leishmania (Jackson et al., 1985). This result may be associated with the different structure of the sporocyst wall of Sarcocystis which may prevent the fluorescein from entering the sporocyst. Although some factors such as the direct action of sunlight, and the competition by other faecal parasites or microflora (Fayer, 1980), have a deleterious effect on the survival of sporocysts in the natural environment, the results of the present study suggest that sporocysts of S. cruzi are able to remain infective for potential intermediate hosts for several months. This period of infectivity is likely to be longer in cooler climates but may be reduced when temperatures greatly fluctuate.
References Bergler, K.G., Erber, H. and Boch, J., 1980. Untersuchungen zur /t)berlebensfiihigkeit von Sporozysten bzw. Oozysten yon Sarcocystis, Toxoplasma, Hammondia und Eimeria unter Laborund Freilandbedingungen. Berl. Mi~nch Tier'~irztl Wochenschr., 93: 288-293. Dubey, J.P., Speer, C.A. and Fayer, R., 1989. Sarcocystosis of Animals and Man. CRC Press, Boca Raton, FL. Ellenby, C., 1968. Desiccation survival in the plant parasitic nematodes, Heterodera rostochiensis Wollenweber and Ditylenchus dipsaci (Kuhn) Filipjev. Proc. R. Soc. Lond. B, 169: 203-213. Fayer, R., 1980. Epidemiology of protozoan infections: the coccidia. Vet. Parasitol., 6: 75-103. Goodrich, H.P., 1944. Coccidian oocysts. Parasitology, 36: 72-79. Heydom, A.O., 1980. Zur Widerstandsfahigkeit von Sarcocystis bovicanis-Sporozysten. Berl. Miinch. Tief~rztl. Wochenschr., 93: 267-270. Jackson, P.R., Pappas, M.G. and Hansen, B.D., 1985. Fluorogenic subs(rate detection of viable intracellular and extracellular pathogenic protozoa. Science, 227: 435-438. Jones, K.H. and Sneft, J.A., 1985. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J. Histochem. Cytochem., 33:77 79. Levine, N.D., 1988. The Protozoan Phylum Apicomplexa. CRC Press, Boca Raton, FL. McKenna, P.B. and Charleston, W.A.G., 1992. The survival of Sarcocystis gigantea sporocysts following exposure to various chemical and physical agents. Vet. Parasitol., 45: 1-16. Parkin, J.T., 1976. The effect of moisture supply upon the development and hatching of the eggs of Nematodirus battus. Parasitology, 73: 353-354. Savini, G., Dunsmore, J.D., Robertson, I.D. and Seneviratna P., 1992. Epidemiology of Sarco(3~stis spp. in cattle of Western Australia. Epidemiol. Infect., 108:107-113. Savini, G., Dunsmore, .I.D. and Robertson, I.D., 1994. Evaluation of a serological test system for the diagnosis of Sarcocystis cruzi infection in cattle using S. cruzi merozoite antigen. Vet. Parasitol., 51:181 189. Schupp, D.G. and Erlandsen, S.L., 1987. A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity. Appl. Envir. Microbiol., 53: 704-707. Wharton, D.A., 1982. The survival of desiccation by the free-living stages of Trichostrongylus colubriJormis (Nematoda: Trichostrongilidae). Parasitology, 84: 455-462. Winston, P.W. and Bates, D., 1960. Saturated solutions for control of humidity in biological research. Ecology, 41: 232-237.