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Effects of low and high temperatures on viability of Parascaris equorum eggs suspended in water
Veterinary Parasitology 142 (2006) 123–128 www.elsevier.com/locate/vetpar
Effects of low and high temperatures on viability of Parascaris equorum eggs suspended in water B. Koudela a,b,*, Sˇ. Bodecˇek c a
Department of Parasitology, University of Veterinary and Pharmaceutical Sciences, Palacke´ho 1-3, 612 42 Brno, Czech Republic b Institute of Parasitology, Biology Centre of Academy of Sciences of the Czech Republic, Branisˇovska´ 31, ˇ eske´ Budeˇjovice, Czech Republic 370 05 C c Equine Clinic, University of Veterinary and Pharmaceutical Sciences, Palacke´ho 1-3, 612 42 Brno, Czech Republic Received 15 November 2005; received in revised form 10 May 2006; accepted 26 May 2006
Abstract Microcentrifuge tubes containing 5000 eggs of Parascaris equorum suspended in water were frozen at 5, 10, 15, 20, and 80 8C for 1–168 h and then thawed at a room temperature. Other samples of P. equorum eggs suspended in water were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 8C incremental temperatures from 40 to 100 8C. At each temperature setting microcentrifuge tubes containing P. equorum eggs were removed 1 and 5 min later. Both, frozen and heated egg suspensions as well as untreated control suspensions were then incubated to test of viability based on the development of infective larvae inside viable eggs. We found out that eggs of P. equorum in water can retain viability and infectivity after freezing and that eggs survive longer at higher freezing temperatures. Our results also indicated that when water containing P. equorum eggs reached temperatures of 60 8C or higher within 1 min, the viability of eggs was lost. # 2006 Elsevier B.V. All rights reserved. Keywords: Viability; Temperature; Parascaris equorum; Eggs
1. Introduction Parascaris equorum is a large roundworm that primarily affects foals and young growing horses. The immunity develops by exposure to these parasites during adolescence, so mature horses are usually not infected by P. equorum. The life cycle of P. equorum starts as the horse swallows eggs containing third-stage larvae with feed, pasture or water. These eggs hatch and the resulting larvae burrow into the wall of the small intestine where they pass through portal blood veins. They migrate to the liver, heart and subsequently to the * Corresponding author. Tel.: +420 541562262; fax: +420 541562266. E-mail address: [email protected] (B. Koudela). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.05.031
lungs. After the migration into airways, the larvae are coughed up, swallowed, return to the small intestine and mature in the duodenum and proximal jejunum. After completion of their migration, the parasites grow in size and first P. equorum eggs appear in the faeces between 72 and 110 days after infection (Lyons et al., 1976; Clayton and Duncan, 1979a,b; Clayton, 1986; Southwood et al., 1998). The infection with P. equorum is associated with poor weight gain, increased total body water content, respiratory disease and colic, impaction, or intussusception. Foals less than 6 months old are most susceptible to the infection and are the source of the greatest egg production. The female of P. equorum lay up to 200,000 eggs per day, and these eggs are passed to the outside in the manure (Clayton, 1986). Clayton and Duncan
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(1979) found around 50 million P. equorum eggs as the highest daily faecal egg output in experimentally infected foal on day 124 after infection. The eggs are very resistant to environmental conditions and may remain infective for years on pastures and in stalls. Because of the longevity of infective P. equorum eggs, the object of control is to prevent any environmental contamination with P. equorum eggs (Clayton, 1986; Southwood et al., 1998). The knowledge of factors affecting the survival and infectivity of eggs of P. equorum is important with respect to the epidemiology and control of infection. However, no study that provided data on survival of P. equorum eggs under environmental conditions can be found in the scientific literature. A review article (Southwood et al., 1998) referred to an unpublished data indicating a loss of infectivity during summer when temperatures were exceeding 39 8C, however provided no information on how that time–temperature relationship was determined. Because of the need to confirm these findings and to provide a series of time– temperature relationships found that render P. equorum eggs non-infectious, the present study was undertaken.
magnetic stirring into an automatic pipette fitted with a disposable tip. The contents were emptied into a 0.5 ml microcentrifuge plastic tube (AXYGEN for PCR, PLab, Czech Republic). 2.2. Low temperature treatment
2. Material and methods
For freezing at 5, 10, 15, 20, and 80 8C, respectively, the following freezers were used: Calex CRM 220-2H, Liebherr CN 36130, Gorenje F247CB, Sanyo Ultra Low Temperature Freezer MDF 792. Although the temperature setting of each freezer was displayed, the actual temperatures inside the glycerol bath were confirmed by a digital thermometer (Testo 110, Lenzkirch, Germany). Microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were placed in a glycerol bath stored overnight in freezers at 5, 10, 15 and 20 8C for 1, 3, 5, 8, 24, and 168 h, and at 80 8C for 1, 3 and 5 h. Four tubes were used for each temperature setting. All microcentrifuge tubes that were removed from freezers were then thawed in the thermostatic bath (32 8C). Four additional microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were held at room temperature as controls during the temperature treatment.
2.1. Parasite
2.3. High temperature treatment
Adult females P. equorum were collected from a mass of parasites accumulated in the stomach of 10-month-old foal with nasogastric reflux and gastrointestinal roundworm impaction. The P. equorum females were dissected and the distal parts of their uteri were cut off and rinsed in tap water. The pieces of uteri were cut in 2–3 mm long sections with a pair of scissors. After that suspension of eggs was pressed trough 177 mm mesh stainless steel sieve and left to settle in water in 2 l glass jar for 1 h. The supernatant was removed and egg material was washed several times in sterile water by centrifugation at 500 rpm for 5 min. The eggs were then placed in 100 ml sterile distilled water with 10 mg ml 1 gentamicin sulfate and were stored in at 4 8C for up to 5 days before being used in the experiments. A working egg solution with approximately 25,000 eggs ml 1 was prepared by suspending the eggs in a sterile distilled water. The standardised number of eggs in a sample was verified under 100 magnification in 10 10 ml samples. The procedure was repeated five times and the tip of the automatic pipette used to dispense the eggs onto the microscopic slide was changed between each replication. A standard sample of 200 ml was taken from the stock egg suspension during
Microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were exposed to temperatures in a DNA thermal cycler with adjustable heated lid (Tpersonal Combi Thermocycler, Whatman/ Biometra, Go¨ttingen, Germany). Twelve temperatures were programmed into the thermal cycler: 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 8C. At each temperature setting eight vials containing P. equorum were each inserted into wells in the heated metal block of the thermal cycler. For each temperature setting, four tubes were removed after 1 min. The four remaining vials were removed after 5 min. After the removal from the wells in the heated block, the vials were held in crushed ice for 2 min. Four additional microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were held at room temperature as controls during the temperature treatment. 2.4. Test of viability Within 15 min after the eggs were treated, each tube was emptied into individual well in a tissue culture plate (24 well, Falcon 3047, Becton Dickinson Labware, NJ) and each well was then supplemented with 1 ml of
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sterile distilled water with 10 mg ml 1 gentamicin sulfate. The plates with P. equorum eggs were incubated for 3 weeks at 25 8C. Aeration was provided by opening the lids and gentle agitation by hand for 3 min five times weekly. The development of eggs was evaluated microscopically (Nikon TMS Inverted Microscope) daily. The morphology of internal structures of P. equorum eggs was also examined and photographed with Olympus AX 70 microscope using Nomarski interference contrast optics (DIC). The criterion for deciding the viability of individual eggs was the presence or absence of viable larvae. For enumeration, a minimum of 200 eggs was counted in each well. The percentage of viable eggs was calculated by dividing the number of viable eggs by the total number of eggs observed and multiplying by 100.
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Fig. 2. Eggs of P. equorum without temperature treatments after 21 days of cultivation. DIC. Bar = 50 mm.
The morphology of P. equorum eggs collected from uteri was different from P. equorum eggs observed in the horse faeces. They were completely without the distinctive brown thick-shell (Fig. 1). After 21 days of cultivation, the majority of control untreated eggs contained motile larvae (Fig. 2), less than 10% of eggs were unfertilised. Eggs frozen at 10, 15, and 20 8C for all periods up to and including 168 h were not morphologically distinguishable from the control samples when examined by DIC microscopy. Eggs frozen at 80 8C appeared morphologically similar whether thawed 1, 8, or 24 h after freezing. When examined by DIC microscopy, some appeared to have no contents, others had partial contents, and still others appeared similar to unfrozen controls. Eggs heated up to and including 55 8C for both periods were also not
morphologically distinguishable from controls when examined by DIC microscopy. Eggs heated at 65 8C and at higher temperature appeared with granular globule in the centre and smaller globules or vacuoles of different sizes at the periphery of eggs (Fig. 3). The results of reduction of viability of P. equorum eggs exposed at defined temperatures are shown in Tables 1 and 2. It was found that 77.3% of the P. equorum eggs are viable after being frozen at 20 8C for 1 week. No significant difference in percent viability was found between eggs frozen at 5, 10, 15, and 20 8C for both periods and untreated control samples. The fact that no viable larvae were found in P. equorum eggs frozen at 80 8C suggested that these eggs were rendered non-infectious. As in all temperature treatments up to 55 8C, the percentage of viable eggs ranged from 78.6 to 87.3%; it is concluded that none of these temperature treatments
Fig. 1. Freshly isolated P. equorum eggs. DIC. Bar = 50 mm.
Fig. 3. Eggs of P. equorum heated at 60 8C for 5 min. DIC. Bar = 50 mm.
3. Results
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Table 1 Effect of low temperature treatments on the viability of P. equorum eggs suspended in watera
Table 2 Effect of high temperature treatments on the viability of P. equorum eggs suspended in watera
Temperature (8C/h)
% viability (standard deviation)
Temperature (8C/min)
% viability (standard deviation)
Control b 5/1 5/3 5/5 5/8 5/24 5/168 10/1 10/3 10/5 10/8 10/24 10/168 15/1 15/3 15/5 15/8 15/24 15/168 20/1 20/3 20/5 20/8 20/24 20/168 80/1 80/3 80/5
92.4 (1.3) 93.4 (2.4) 92.0 (1.3) 92.4 (2.7) 89.4 (3.9) 90.8 (3.3) 86.9 (5.7) 93.0 (1.2) 91.9 (3.3) 89.2 (3.7) 90.2 (2.5) 86.8 (6.2) 85.9 (4.1) 87.2 (3.2) 88.9 (2.0) 85.9 (3.3) 86.5 (4.2) 89.3 (2.6) 83.2 (3.6) 88.2 (2.3) 87.5 (3.0) 86.0 (4.2) 87.1 (4.3) 86.7 (3.8) 77.3 (8.4) 0 (0) 0 (0) 0 (0)
Controlb 40/1 40/5 45/1 45/5 50/1 50/5 55/1 55/5 60/1 60/5 65/1 65/5 70/1 70/5 75/1 75/5 80/1 80/5 85/1 85/5 90/1 90/5 95/1 95/5 100/1 100/5
92.4 (1.3) 87.3 (2.3) 85.2 (4.0) 86.9 (3.5) 84.0 (2.8) 85.0 (3.3) 83.5 (5.5) 86.3 (3.3) 78.6 (9.7) 38.2 (16.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
ND: not determined. a The value reported for percent viability is a mean of five replicate samples for each of microcentrifuge tube/well. b Control samples incubated at room temperature.
affected the viability of the P. equorum eggs. A significant decline in the percentage of viable eggs was observed only among the eggs heated at 60 8C. In the samples heated at 60 8C for 1 min, the percentage of viable P. equorum eggs decreased to 38.2%. In the samples that were treated at 60 8C for 5 min no viable eggs were observed. These data indicate that to achieve a negligible level of viable P. equorum eggs suspended in water it requires a heating of 60 8C for 5 min. 4. Discussion The techniques for assessing the viability of ascarid eggs are based on the development of infective larvae inside viable eggs when the sample is incubated in an antimicrobial solution that prevents the growth of other organisms that may interfere with egg development (Gaspard et al., 1996; Gaasenbeek and Borgsteede, 1998; Johnson et al., 1998). Some of the antimicrobial solutions appear to have a negative impact on the
a
The value reported for percent viability is a mean of five replicate samples for each of microcentrifuge tube/well. b Control samples incubated at room temperature.
viability of the eggs. It has been reported that eggs incubated in 1% formalin showed a retarded development compared to those incubated in water or 0.1N H2SO4 (Oksanen et al., 1990). Regardless of no decrease in the viability of A. suum eggs incubated in 0.1N H2SO4 (Nelson and Darby, 2001) we incubated P. equorum eggs in sterile distilled water supplemented with gentamicin sulfate to prevent a bacterial contamination of eggs suspension. The present study did not quantitate the different larval stages emerging inside eggs after different periods of cultivation. According to Clayton and Duncan (1979b) a period of 10 days of cultivation at 27 8C is needed before P. equorum eggs become infective to horses. This disagrees with our observation that the first apparently fully embryonated eggs were seen in the control sample 19 days after the start of cultivation. However, our results are consistent with previously published observations that the rate of development to the infective stage is slower when ascarid egg density is high (Eriksen, 1990).
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In the present study, when an aqueous suspension of P. equorum eggs free of extraneous biological debris was used, embryonation remained high when eggs were exposed to freezing temperatures. The finding of viable P. equorum eggs in suspensions that were frozen at 10, 15, and 20 8C is new and of an epidemiological importance. Therefore, on the basis of the findings from the present study it cannot be assumed that water containing P. equorum eggs is non-infectious by being frozen. Furthermore, because P. equorum eggs appeared to survive relatively long at freezing temperatures, the survival of P. equorum eggs on the pasture might be extended beyond the times observed in the present study when water containing minerals or organic material is frozen at temperature above 10 8C. Such conditions might be found in areas where, after ground temperatures fall to just below zero enabling P. equorum to survive perhaps for weeks or months. The influence of temperatures on the viability of ascarid eggs is rarely reported. Arene (1986) studied the influence of embryonation temperature on the viability on A. suum eggs and found the maximum rate of egg development at 31 8C. O’Lorcain (1995) studied the effect of freezing on Toxocara canis and Toxocara cati eggs inactivation. He placed T. canis and T. cati embryonated eggs in a freezer compartment of domestic refrigerator and maintained the suspensions of eggs at the average temperature 12.76 8C (minimum 20 8C, maximum 7 8C) over a period of 34 days. It was observed that the longer the eggs were frozen the less viable they became. However, a quarter of both T. canis and T. cati eggs survived after the 34th day of freezing. O’Lorcain (1995) also found that T. cati embryonated eggs were more capable to survive prolonged exposure to freezing temperature than T. canis. In the samples treated with high temperatures, the viable P. equorum eggs were still occurred when they were exposed to temperature as high as 60 8C with a heat-up time of 1 min. However, when eggs were exposed to temperatures of 60 8C with a heat-up time of 5 min, viability could not be detected in any samples. These findings indicate that P. equorum eggs in water can be rendered non-infectious when held for a relatively short time at temperatures that reached 60 8C. The conditions used for testing in the present study eliminated the potentially limitless number of variables that might be expected in water that contains mineral or vegetable sediment. Other complex material, such as horse faeces, manure or sludge might also yield results different from those obtained in the present study.
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The ascarid eggs are highly resistant and they can survive in a variety of settings including aerobic and anaerobic environments. The literature contains many studies on the time–temperature survival of A. lumbricoides and A. suum eggs under different environmental conditions. Under the absence of other factors, temperatures less than 40 8C are usually not effective at inactivating Ascaris eggs except over very long time periods (>1 year). During a biological degradation, such as an anaerobic or aerobic digestion, many factors may affect the degree of ascarid egg inactivation, however, temperature is still the dominant factor (Feachem et al., 1983). Thus, at mesophilic temperatures (<40 8C), both anaerobic and aerobic environments are only partially effective at inactivating Ascaris eggs, whereas the thermophilic temperatures (>50 8C) can inactivate eggs to under the detectable limits (Pecson and Nelson, 2005). However, on the basis of the findings from the present study it cannot be unambiguously assumed that water containing P. equorum eggs is rendered non-infectious by being exposed to thermophilic temperatures. In order to ensure total killing of P. equorum eggs in the faeces or manure, further research is needed to quantify the inactivating effect of other factors. Field models will also need to take diurnal temperature fluctuations into account. Acknowledgement The financial support from the Grant Agency of the Czech Republic (grant no. 524/03/H133) is gratefully acknowledged. References Arene, F.O.I., 1986. Ascaris suum: influence of embryonation temperature on the viability of the infective larva. J. Therm. Biol. 11, 9–15. Clayton, H.M., 1986. Ascarids. Recent advances. Vet. Clin. North. Am. Equine Pract. 2, 313–328. Clayton, H.M., Duncan, J.L., 1979a. The migration and development of Parascaris equorum in the horse. Int. J. Parasitol. 9, 285–292. Clayton, H.M., Duncan, J.L., 1979b. The development of immunity to Parascaris equorum infection in the foal. Res. Vet. Sci. 26, 383– 384. Eriksen, L., 1990. Ascaris suum: influence of egg density on in vitro development from embryonated egg to infective stage. Acta Vet. Scand. 31, 489–491. Feachem, R.G., Bradley, D.J., Garelick, H., Mara, D.D., 1983. Sanitation and Disease: Health Aspects of Excreta and Wastewater Management. New York, John Wiley and Sons. Gaasenbeek, C.P.H., Borgsteede, F.H.M., 1998. Studies on the survival of Ascaris suum eggs under laboratory and simulated field conditions. Vet. Parasitol. 75, 227–234.
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Gaspard, P., Wiart, J., Schwartzbrod, J., 1996. A method for assessing the viability of nematode eggs in sludge. Environ. Technol. 17, 415–420. Johnson, P.W., Dixon, R., Ross, A.D., 1998. An in-vitro test for assessing the viability of Ascaris suum eggs exposed to various sewage treatment processes. Int. J. Parasitol. 28, 627–633. Lyons, E.T., Drudge, J.H., Tolliver, S.C., 1976. Studies on the development and chemotherapy of larvae of Parascaris equorum (Nematoda: Ascaridoidea) in experimentally and naturally infected foals. J. Parasitol. 62, 453–459. Nelson, K.L., Darby, J.L., 2001. Inactivation of viable Ascaris eggs by reagents during enumeration. Appl. Environ. Microbiol. 67, 5453– 5459.
Oksanen, A., Eriksen, L., Roepstorff, A., Ilsoe, B., Nansen, P., Lind, P., 1990. Embryonation and infectivity of Ascaris suum eggs. A comparison of eggs collected from worm uteri with eggs isolated from pig faeces. Acta Vet. Scand. 31, 393–398. O’Lorcain, P., 1995. The effects of freezing on the viability of Toxocara canis and T. cati embryonated eggs. J. Helminthol. 69, 169–171. Pecson, B.M., Nelson, K.L., 2005. Inactivation of Ascaris suum Eggs by Ammonia. Environ. Sci. Technol. 39, 7909–7914. Southwood, L.L., Baxter, G.M., Bennett, D.G., Ragle, C.A., 1998. Ascarid impaction in young horses. Compend. Contin. Educ. Pract. Vet. 20, 100–106.
Effects of low and high temperatures on viability of Parascaris equorum eggs suspended in water B. Koudela a,b,*, Sˇ. Bodecˇek c a
Department of Parasitology, University of Veterinary and Pharmaceutical Sciences, Palacke´ho 1-3, 612 42 Brno, Czech Republic b Institute of Parasitology, Biology Centre of Academy of Sciences of the Czech Republic, Branisˇovska´ 31, ˇ eske´ Budeˇjovice, Czech Republic 370 05 C c Equine Clinic, University of Veterinary and Pharmaceutical Sciences, Palacke´ho 1-3, 612 42 Brno, Czech Republic Received 15 November 2005; received in revised form 10 May 2006; accepted 26 May 2006
Abstract Microcentrifuge tubes containing 5000 eggs of Parascaris equorum suspended in water were frozen at 5, 10, 15, 20, and 80 8C for 1–168 h and then thawed at a room temperature. Other samples of P. equorum eggs suspended in water were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 8C incremental temperatures from 40 to 100 8C. At each temperature setting microcentrifuge tubes containing P. equorum eggs were removed 1 and 5 min later. Both, frozen and heated egg suspensions as well as untreated control suspensions were then incubated to test of viability based on the development of infective larvae inside viable eggs. We found out that eggs of P. equorum in water can retain viability and infectivity after freezing and that eggs survive longer at higher freezing temperatures. Our results also indicated that when water containing P. equorum eggs reached temperatures of 60 8C or higher within 1 min, the viability of eggs was lost. # 2006 Elsevier B.V. All rights reserved. Keywords: Viability; Temperature; Parascaris equorum; Eggs
1. Introduction Parascaris equorum is a large roundworm that primarily affects foals and young growing horses. The immunity develops by exposure to these parasites during adolescence, so mature horses are usually not infected by P. equorum. The life cycle of P. equorum starts as the horse swallows eggs containing third-stage larvae with feed, pasture or water. These eggs hatch and the resulting larvae burrow into the wall of the small intestine where they pass through portal blood veins. They migrate to the liver, heart and subsequently to the * Corresponding author. Tel.: +420 541562262; fax: +420 541562266. E-mail address: [email protected] (B. Koudela). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.05.031
lungs. After the migration into airways, the larvae are coughed up, swallowed, return to the small intestine and mature in the duodenum and proximal jejunum. After completion of their migration, the parasites grow in size and first P. equorum eggs appear in the faeces between 72 and 110 days after infection (Lyons et al., 1976; Clayton and Duncan, 1979a,b; Clayton, 1986; Southwood et al., 1998). The infection with P. equorum is associated with poor weight gain, increased total body water content, respiratory disease and colic, impaction, or intussusception. Foals less than 6 months old are most susceptible to the infection and are the source of the greatest egg production. The female of P. equorum lay up to 200,000 eggs per day, and these eggs are passed to the outside in the manure (Clayton, 1986). Clayton and Duncan
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(1979) found around 50 million P. equorum eggs as the highest daily faecal egg output in experimentally infected foal on day 124 after infection. The eggs are very resistant to environmental conditions and may remain infective for years on pastures and in stalls. Because of the longevity of infective P. equorum eggs, the object of control is to prevent any environmental contamination with P. equorum eggs (Clayton, 1986; Southwood et al., 1998). The knowledge of factors affecting the survival and infectivity of eggs of P. equorum is important with respect to the epidemiology and control of infection. However, no study that provided data on survival of P. equorum eggs under environmental conditions can be found in the scientific literature. A review article (Southwood et al., 1998) referred to an unpublished data indicating a loss of infectivity during summer when temperatures were exceeding 39 8C, however provided no information on how that time–temperature relationship was determined. Because of the need to confirm these findings and to provide a series of time– temperature relationships found that render P. equorum eggs non-infectious, the present study was undertaken.
magnetic stirring into an automatic pipette fitted with a disposable tip. The contents were emptied into a 0.5 ml microcentrifuge plastic tube (AXYGEN for PCR, PLab, Czech Republic). 2.2. Low temperature treatment
2. Material and methods
For freezing at 5, 10, 15, 20, and 80 8C, respectively, the following freezers were used: Calex CRM 220-2H, Liebherr CN 36130, Gorenje F247CB, Sanyo Ultra Low Temperature Freezer MDF 792. Although the temperature setting of each freezer was displayed, the actual temperatures inside the glycerol bath were confirmed by a digital thermometer (Testo 110, Lenzkirch, Germany). Microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were placed in a glycerol bath stored overnight in freezers at 5, 10, 15 and 20 8C for 1, 3, 5, 8, 24, and 168 h, and at 80 8C for 1, 3 and 5 h. Four tubes were used for each temperature setting. All microcentrifuge tubes that were removed from freezers were then thawed in the thermostatic bath (32 8C). Four additional microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were held at room temperature as controls during the temperature treatment.
2.1. Parasite
2.3. High temperature treatment
Adult females P. equorum were collected from a mass of parasites accumulated in the stomach of 10-month-old foal with nasogastric reflux and gastrointestinal roundworm impaction. The P. equorum females were dissected and the distal parts of their uteri were cut off and rinsed in tap water. The pieces of uteri were cut in 2–3 mm long sections with a pair of scissors. After that suspension of eggs was pressed trough 177 mm mesh stainless steel sieve and left to settle in water in 2 l glass jar for 1 h. The supernatant was removed and egg material was washed several times in sterile water by centrifugation at 500 rpm for 5 min. The eggs were then placed in 100 ml sterile distilled water with 10 mg ml 1 gentamicin sulfate and were stored in at 4 8C for up to 5 days before being used in the experiments. A working egg solution with approximately 25,000 eggs ml 1 was prepared by suspending the eggs in a sterile distilled water. The standardised number of eggs in a sample was verified under 100 magnification in 10 10 ml samples. The procedure was repeated five times and the tip of the automatic pipette used to dispense the eggs onto the microscopic slide was changed between each replication. A standard sample of 200 ml was taken from the stock egg suspension during
Microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were exposed to temperatures in a DNA thermal cycler with adjustable heated lid (Tpersonal Combi Thermocycler, Whatman/ Biometra, Go¨ttingen, Germany). Twelve temperatures were programmed into the thermal cycler: 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 8C. At each temperature setting eight vials containing P. equorum were each inserted into wells in the heated metal block of the thermal cycler. For each temperature setting, four tubes were removed after 1 min. The four remaining vials were removed after 5 min. After the removal from the wells in the heated block, the vials were held in crushed ice for 2 min. Four additional microcentrifuge tubes containing 5000 eggs of P. equorum in 200 ml of distilled water were held at room temperature as controls during the temperature treatment. 2.4. Test of viability Within 15 min after the eggs were treated, each tube was emptied into individual well in a tissue culture plate (24 well, Falcon 3047, Becton Dickinson Labware, NJ) and each well was then supplemented with 1 ml of
B. Koudela, Sˇ. Bodecˇek / Veterinary Parasitology 142 (2006) 123–128
sterile distilled water with 10 mg ml 1 gentamicin sulfate. The plates with P. equorum eggs were incubated for 3 weeks at 25 8C. Aeration was provided by opening the lids and gentle agitation by hand for 3 min five times weekly. The development of eggs was evaluated microscopically (Nikon TMS Inverted Microscope) daily. The morphology of internal structures of P. equorum eggs was also examined and photographed with Olympus AX 70 microscope using Nomarski interference contrast optics (DIC). The criterion for deciding the viability of individual eggs was the presence or absence of viable larvae. For enumeration, a minimum of 200 eggs was counted in each well. The percentage of viable eggs was calculated by dividing the number of viable eggs by the total number of eggs observed and multiplying by 100.
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Fig. 2. Eggs of P. equorum without temperature treatments after 21 days of cultivation. DIC. Bar = 50 mm.
The morphology of P. equorum eggs collected from uteri was different from P. equorum eggs observed in the horse faeces. They were completely without the distinctive brown thick-shell (Fig. 1). After 21 days of cultivation, the majority of control untreated eggs contained motile larvae (Fig. 2), less than 10% of eggs were unfertilised. Eggs frozen at 10, 15, and 20 8C for all periods up to and including 168 h were not morphologically distinguishable from the control samples when examined by DIC microscopy. Eggs frozen at 80 8C appeared morphologically similar whether thawed 1, 8, or 24 h after freezing. When examined by DIC microscopy, some appeared to have no contents, others had partial contents, and still others appeared similar to unfrozen controls. Eggs heated up to and including 55 8C for both periods were also not
morphologically distinguishable from controls when examined by DIC microscopy. Eggs heated at 65 8C and at higher temperature appeared with granular globule in the centre and smaller globules or vacuoles of different sizes at the periphery of eggs (Fig. 3). The results of reduction of viability of P. equorum eggs exposed at defined temperatures are shown in Tables 1 and 2. It was found that 77.3% of the P. equorum eggs are viable after being frozen at 20 8C for 1 week. No significant difference in percent viability was found between eggs frozen at 5, 10, 15, and 20 8C for both periods and untreated control samples. The fact that no viable larvae were found in P. equorum eggs frozen at 80 8C suggested that these eggs were rendered non-infectious. As in all temperature treatments up to 55 8C, the percentage of viable eggs ranged from 78.6 to 87.3%; it is concluded that none of these temperature treatments
Fig. 1. Freshly isolated P. equorum eggs. DIC. Bar = 50 mm.
Fig. 3. Eggs of P. equorum heated at 60 8C for 5 min. DIC. Bar = 50 mm.
3. Results
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Table 1 Effect of low temperature treatments on the viability of P. equorum eggs suspended in watera
Table 2 Effect of high temperature treatments on the viability of P. equorum eggs suspended in watera
Temperature (8C/h)
% viability (standard deviation)
Temperature (8C/min)
% viability (standard deviation)
Control b 5/1 5/3 5/5 5/8 5/24 5/168 10/1 10/3 10/5 10/8 10/24 10/168 15/1 15/3 15/5 15/8 15/24 15/168 20/1 20/3 20/5 20/8 20/24 20/168 80/1 80/3 80/5
92.4 (1.3) 93.4 (2.4) 92.0 (1.3) 92.4 (2.7) 89.4 (3.9) 90.8 (3.3) 86.9 (5.7) 93.0 (1.2) 91.9 (3.3) 89.2 (3.7) 90.2 (2.5) 86.8 (6.2) 85.9 (4.1) 87.2 (3.2) 88.9 (2.0) 85.9 (3.3) 86.5 (4.2) 89.3 (2.6) 83.2 (3.6) 88.2 (2.3) 87.5 (3.0) 86.0 (4.2) 87.1 (4.3) 86.7 (3.8) 77.3 (8.4) 0 (0) 0 (0) 0 (0)
Controlb 40/1 40/5 45/1 45/5 50/1 50/5 55/1 55/5 60/1 60/5 65/1 65/5 70/1 70/5 75/1 75/5 80/1 80/5 85/1 85/5 90/1 90/5 95/1 95/5 100/1 100/5
92.4 (1.3) 87.3 (2.3) 85.2 (4.0) 86.9 (3.5) 84.0 (2.8) 85.0 (3.3) 83.5 (5.5) 86.3 (3.3) 78.6 (9.7) 38.2 (16.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
ND: not determined. a The value reported for percent viability is a mean of five replicate samples for each of microcentrifuge tube/well. b Control samples incubated at room temperature.
affected the viability of the P. equorum eggs. A significant decline in the percentage of viable eggs was observed only among the eggs heated at 60 8C. In the samples heated at 60 8C for 1 min, the percentage of viable P. equorum eggs decreased to 38.2%. In the samples that were treated at 60 8C for 5 min no viable eggs were observed. These data indicate that to achieve a negligible level of viable P. equorum eggs suspended in water it requires a heating of 60 8C for 5 min. 4. Discussion The techniques for assessing the viability of ascarid eggs are based on the development of infective larvae inside viable eggs when the sample is incubated in an antimicrobial solution that prevents the growth of other organisms that may interfere with egg development (Gaspard et al., 1996; Gaasenbeek and Borgsteede, 1998; Johnson et al., 1998). Some of the antimicrobial solutions appear to have a negative impact on the
a
The value reported for percent viability is a mean of five replicate samples for each of microcentrifuge tube/well. b Control samples incubated at room temperature.
viability of the eggs. It has been reported that eggs incubated in 1% formalin showed a retarded development compared to those incubated in water or 0.1N H2SO4 (Oksanen et al., 1990). Regardless of no decrease in the viability of A. suum eggs incubated in 0.1N H2SO4 (Nelson and Darby, 2001) we incubated P. equorum eggs in sterile distilled water supplemented with gentamicin sulfate to prevent a bacterial contamination of eggs suspension. The present study did not quantitate the different larval stages emerging inside eggs after different periods of cultivation. According to Clayton and Duncan (1979b) a period of 10 days of cultivation at 27 8C is needed before P. equorum eggs become infective to horses. This disagrees with our observation that the first apparently fully embryonated eggs were seen in the control sample 19 days after the start of cultivation. However, our results are consistent with previously published observations that the rate of development to the infective stage is slower when ascarid egg density is high (Eriksen, 1990).
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In the present study, when an aqueous suspension of P. equorum eggs free of extraneous biological debris was used, embryonation remained high when eggs were exposed to freezing temperatures. The finding of viable P. equorum eggs in suspensions that were frozen at 10, 15, and 20 8C is new and of an epidemiological importance. Therefore, on the basis of the findings from the present study it cannot be assumed that water containing P. equorum eggs is non-infectious by being frozen. Furthermore, because P. equorum eggs appeared to survive relatively long at freezing temperatures, the survival of P. equorum eggs on the pasture might be extended beyond the times observed in the present study when water containing minerals or organic material is frozen at temperature above 10 8C. Such conditions might be found in areas where, after ground temperatures fall to just below zero enabling P. equorum to survive perhaps for weeks or months. The influence of temperatures on the viability of ascarid eggs is rarely reported. Arene (1986) studied the influence of embryonation temperature on the viability on A. suum eggs and found the maximum rate of egg development at 31 8C. O’Lorcain (1995) studied the effect of freezing on Toxocara canis and Toxocara cati eggs inactivation. He placed T. canis and T. cati embryonated eggs in a freezer compartment of domestic refrigerator and maintained the suspensions of eggs at the average temperature 12.76 8C (minimum 20 8C, maximum 7 8C) over a period of 34 days. It was observed that the longer the eggs were frozen the less viable they became. However, a quarter of both T. canis and T. cati eggs survived after the 34th day of freezing. O’Lorcain (1995) also found that T. cati embryonated eggs were more capable to survive prolonged exposure to freezing temperature than T. canis. In the samples treated with high temperatures, the viable P. equorum eggs were still occurred when they were exposed to temperature as high as 60 8C with a heat-up time of 1 min. However, when eggs were exposed to temperatures of 60 8C with a heat-up time of 5 min, viability could not be detected in any samples. These findings indicate that P. equorum eggs in water can be rendered non-infectious when held for a relatively short time at temperatures that reached 60 8C. The conditions used for testing in the present study eliminated the potentially limitless number of variables that might be expected in water that contains mineral or vegetable sediment. Other complex material, such as horse faeces, manure or sludge might also yield results different from those obtained in the present study.
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The ascarid eggs are highly resistant and they can survive in a variety of settings including aerobic and anaerobic environments. The literature contains many studies on the time–temperature survival of A. lumbricoides and A. suum eggs under different environmental conditions. Under the absence of other factors, temperatures less than 40 8C are usually not effective at inactivating Ascaris eggs except over very long time periods (>1 year). During a biological degradation, such as an anaerobic or aerobic digestion, many factors may affect the degree of ascarid egg inactivation, however, temperature is still the dominant factor (Feachem et al., 1983). Thus, at mesophilic temperatures (<40 8C), both anaerobic and aerobic environments are only partially effective at inactivating Ascaris eggs, whereas the thermophilic temperatures (>50 8C) can inactivate eggs to under the detectable limits (Pecson and Nelson, 2005). However, on the basis of the findings from the present study it cannot be unambiguously assumed that water containing P. equorum eggs is rendered non-infectious by being exposed to thermophilic temperatures. In order to ensure total killing of P. equorum eggs in the faeces or manure, further research is needed to quantify the inactivating effect of other factors. Field models will also need to take diurnal temperature fluctuations into account. Acknowledgement The financial support from the Grant Agency of the Czech Republic (grant no. 524/03/H133) is gratefully acknowledged. References Arene, F.O.I., 1986. Ascaris suum: influence of embryonation temperature on the viability of the infective larva. J. Therm. Biol. 11, 9–15. Clayton, H.M., 1986. Ascarids. Recent advances. Vet. Clin. North. Am. Equine Pract. 2, 313–328. Clayton, H.M., Duncan, J.L., 1979a. The migration and development of Parascaris equorum in the horse. Int. J. Parasitol. 9, 285–292. Clayton, H.M., Duncan, J.L., 1979b. The development of immunity to Parascaris equorum infection in the foal. Res. Vet. Sci. 26, 383– 384. Eriksen, L., 1990. Ascaris suum: influence of egg density on in vitro development from embryonated egg to infective stage. Acta Vet. Scand. 31, 489–491. Feachem, R.G., Bradley, D.J., Garelick, H., Mara, D.D., 1983. Sanitation and Disease: Health Aspects of Excreta and Wastewater Management. New York, John Wiley and Sons. Gaasenbeek, C.P.H., Borgsteede, F.H.M., 1998. Studies on the survival of Ascaris suum eggs under laboratory and simulated field conditions. Vet. Parasitol. 75, 227–234.
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Gaspard, P., Wiart, J., Schwartzbrod, J., 1996. A method for assessing the viability of nematode eggs in sludge. Environ. Technol. 17, 415–420. Johnson, P.W., Dixon, R., Ross, A.D., 1998. An in-vitro test for assessing the viability of Ascaris suum eggs exposed to various sewage treatment processes. Int. J. Parasitol. 28, 627–633. Lyons, E.T., Drudge, J.H., Tolliver, S.C., 1976. Studies on the development and chemotherapy of larvae of Parascaris equorum (Nematoda: Ascaridoidea) in experimentally and naturally infected foals. J. Parasitol. 62, 453–459. Nelson, K.L., Darby, J.L., 2001. Inactivation of viable Ascaris eggs by reagents during enumeration. Appl. Environ. Microbiol. 67, 5453– 5459.
Oksanen, A., Eriksen, L., Roepstorff, A., Ilsoe, B., Nansen, P., Lind, P., 1990. Embryonation and infectivity of Ascaris suum eggs. A comparison of eggs collected from worm uteri with eggs isolated from pig faeces. Acta Vet. Scand. 31, 393–398. O’Lorcain, P., 1995. The effects of freezing on the viability of Toxocara canis and T. cati embryonated eggs. J. Helminthol. 69, 169–171. Pecson, B.M., Nelson, K.L., 2005. Inactivation of Ascaris suum Eggs by Ammonia. Environ. Sci. Technol. 39, 7909–7914. Southwood, L.L., Baxter, G.M., Bennett, D.G., Ragle, C.A., 1998. Ascarid impaction in young horses. Compend. Contin. Educ. Pract. Vet. 20, 100–106.