Parasitol Res (2016) 115:863–866 DOI 10.1007/s00436-015-4813-4
SHORT COMMUNICATION
Inactivation of Cryptosporidium parvum under laboratory conditions Cora Delling 1 & Ivette Holzhausen 1 & Arwid Daugschies 1 & Matthias Lendner 1
Received: 12 October 2015 / Accepted: 2 November 2015 / Published online: 14 November 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The aim of the present study was to evaluate alternatives for inactivating Cryptosporidium parvum under experimental conditions. Disinfectants against this protozoan are usually based on cresols and often difficult to handle in laboratories. Four different substances (ethanol, denatured ethanol, sodium hypochlorite and peroxide) at different concentrations were tested for several exposure times (30 min, 2 h, 4 h, 12 h and 24 h). The results show an inactivation over 99 % by using 10 % H2O2 at an exposure time over 2 h as well as 3 and 6 % NaOCl after 12 h of exposure. Furthermore, the ability of UVC light to inactivate oocysts on smooth surfaces (e.g., laminar flow) was evaluated. To mimic laboratory conditions, oocysts were given on germ carriers. Best results (>99 %) were achieved at an exposure time of 30 min (100.8 mJ/cm2). Keywords Cryptosporidium . Inactivation . UV-C light . Germ carrier Cryptosporidia are important disease agents in humans and animals and may be transmitted from man to man or as zoonotic infection (Lendner et al. 2011). Immunodeficient humans and newborns are especially susceptible and manifest heavy clinical symptoms up to lethal forms of the infection. A main problem in combating the parasite is its resistance against a lot of disinfectants and environmental influences. Inactivation is defined as a reduction of infectious parasite stages. Due to the resistance of the oocysts, a reduction of Cora Delling and Ivette Holzhausen contributed equally to this work. * Matthias Lendner [email protected] 1
Institute of Parasitology, University Leipzig, Leipzig, Germany
infectious parasites by 2 log10 levels is already a good result (Shahiduzzaman et al. 2009). The approved disinfectants are mostly based on chlor-m-cresol which is corrosive and irritating to mucosa. Therefore, cresols are not suitable for the inactivation of oocysts under experimental conditions, e.g. under a laminar flow or in tubings of FACS machines, which makes it difficult to work with this parasites considering that full inactivation has to be achieved following laboratory experimentation. There are many studies dealing with the inactivation of Cryptosporidium parvum oocysts by several substances. Ethanol (Weir et al. 2002), sodium hypochlorite (Fayer 1995; Barbee et al. 1999; Weir et al. 2002; Coulliette et al. 2006) and peroxide (Barbee et al. 1999; Weir et al. 2002; Castro-Hermida et al. 2006) were already tested, but often, exposure times were quiet short and a long course of different exposure times was not undertaken so far. In the present study, such long-term exposure times have been examined. Additionally, the possibility to use UV-C light for inactivating the protozoan was tested. Until now, several studies have shown the effectiveness of UV light to inactivate C. parvum oocysts but most publications deal with the treatment of drinking water (Morita et al. 2002; Clancy et al. 2004) or apple cider (Hanes et al. 2002). While most studies use suspended oocysts, we report on the UV-C light inactivation on smooth surfaces using a germ carrier assay developed to test disinfectants (Dresely et al. 2015). The aim of the study was to find appropriate alternatives regarding inactivation of C. parvum oocysts that can be used under laboratory conditions and are less aggressive than cresols. For both experiments, an in-house strain of C. parvum was used. The maintenance of the parasites was assured through passages in neonatal calves every 3 months. Oocysts were isolated and stored as described earlier (Najdrowski et al. 2007; Dresely et al. 2015). To test alternative disinfectants, 4×105 HCT-8 cells were seeded in 24-well plates with 1 ml growth medium (RPMI 1640, 5 % foetal calf serum (FCS),
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Parasitol Res (2016) 115:863–866
1 % sodium pyruvate) 24 h before inactivation and infection. C. parvum oocysts were centrifuged for 5 min at 7000g, washed with sterile phosphate-buffered saline (PBS) and resuspended in 1 ml water of standardized hardness for counting in a Neubauer chamber. Oocysts at 3×106 per tube were treated with the test substances. Every test was performed in triplicate. After the treatment, oocysts were centrifuged for 7 min at 15,000g and washed with sterile PBS twice. The oocyst pellet was resuspended in 1 ml growth medium. HCT-8 cells were infected with 2×105 oocysts in the presence of sodium taurocholate (0.2 %), amphotericin B (1 %) and gentamycin (0.5 %). Heat-inactivated oocysts (70 °C for 20 min) were used as positive control, untreated oocysts as negative control and non-infected cells as no template control. After 3 h of excystation (37 °C, 5 % CO2), the cells were washed twice with PBS and provided with new growth medium (supplemented with amphotericin B (1 %) and gentamycin (0.5 %)). After 24 h, the cells were harvested, resuspended in 200 μl PBS and stored at −20 °C until DNA extraction. The DNA extraction was performed with a QIAamp DNA Mini Kit according to the manufacturer’s protocol. The DNA concentration was measured and adjusted to 80 ng/μl. A real-time PCR w a s p er f o r m e d u s i n g t he p r o t o c o l d e s c r i be d b y Shahiduzzaman et al. (2009) with some modifications. The Maxima Probe (Thermo Scientific™) master mix was used, and a standardized amount of 400 ng DNA was applied to each reaction. Each run was performed in technical duplicates. Inactivation using UV-C light was performed in a safety cabinet (HERA SAFE KS 12) with two integrated UV-C ultraviolet lamps (Osram HNS 15W OFR) at both sides emitting light at 254 nm. The UV-C intensity was measured at different points of the working area with a radiation meter (PCE-UV36, PCE Instruments UK Ltd). Inactivation was performed in the centre of the working area. Table 1 Inactivation of C. parvum oocysts by different substances
Treatment
Heat inactivateda Ethanol denatured 100 % ethanol 70 % ethanol 10 % H2O2 3 % H2O2 1.5 % NaOCl 3 % NaOCl 6 % NaOCl
% inactivation±SD 30 min
2h
4h
12 h
24 h
99.96±0.02 97.91±0.22 96.17±0.80 77.80±4.49 97.29±0.14 93.85±3.28 −37.64±6.01 54.32±17.98 88.19±3.43
99.97±0.01 99.21±0.57b 90.41±3.70 96.79±0.55 99.44±0.62c 97.89±0.79 16.17±22.73 95.86±0.81 95.61±1.69
99.94±0.06 98.18±1.54c 91.56±5.51 94.78±1.72 99.68±0.39c 98.31±1.52 89.73±4.02 94.46±1.36 97.84±0.68
99.92±0.06 98.63±0.19 93.89±5.76 84.26±10.40 99.31±0.63c 97.45±1.46 94.53±1.03 99.63±0.32c 99.75±0.20c
99.99±0.02 98.97±0.23 86.24±1.15 88.55±1.47 99.75±0.08 95.76±1.21 97.21±1.48 99.63±0.08 99.91±0.15
n=3 a
To evaluate inactivation by UV-C light, a germ carrier assay as described previously was used (Dresely et al. 2015). Briefly, 1×107 oocysts in 50 μl PBS with 0.03 % bovine serum albumin (BSA) were spread on a 2-cm brushed stainless steel disc and were dried for 30 min in a laminar flow. Subsequently, germ carriers were exposed to UV-C light for 10, 20 and 30 min. To recover the oocysts, germ carriers were transferred into 50-ml Falcon tubes containing 5 ml of distilled water. After 2 min, the Falcon tubes were vortexed for 1 min. The germ carriers were removed and the Falcon tubes were centrifuged at 1000g for 8 min. Infection, harvesting cells and further treatment were done as described above. To test the ability of common chemicals to inactivate C. parvum oocysts, a suspension assay was used. The most promising substances and exposure times were tested twice in triplicate to ensure reproducibility. Heat-inactivated oocysts were used as positive control and exceeded 99.9 % inactivation in all experiments. Highest inactivation (over 99 %) was achieved using 10 % H2O2 at an exposure time of over 2 h as well as 3 and 6 % NaOCl for more than 12 h. All results are summarized in Table 1. Ten percent H2O2 seems to be the best choice as an alternative to cresols regarding inactivation of C. parvum oocysts under experimental conditions, e.g. as a positive control in suspension tests. The results show good agreement with former studies regarding the effectiveness of hydrogen peroxide in cell culture. H2O2 (6 %) was described as an effective disinfectant against C. parvum oocysts (Weir et al. 2002), being able to reduce the oocysts’ infectivity greater than 3 log10 (Barbee et al. 1999). Similarly, Castro-Hermida et al. (2006) tested the commercial disinfectant AGROXIDE II (Laboratoires CEETAL) diluted to 10 % (vol/vol), with the major component being H2O2, in neonatal mice and reported a significantly lower infection (P
SHORT COMMUNICATION
Inactivation of Cryptosporidium parvum under laboratory conditions Cora Delling 1 & Ivette Holzhausen 1 & Arwid Daugschies 1 & Matthias Lendner 1
Received: 12 October 2015 / Accepted: 2 November 2015 / Published online: 14 November 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The aim of the present study was to evaluate alternatives for inactivating Cryptosporidium parvum under experimental conditions. Disinfectants against this protozoan are usually based on cresols and often difficult to handle in laboratories. Four different substances (ethanol, denatured ethanol, sodium hypochlorite and peroxide) at different concentrations were tested for several exposure times (30 min, 2 h, 4 h, 12 h and 24 h). The results show an inactivation over 99 % by using 10 % H2O2 at an exposure time over 2 h as well as 3 and 6 % NaOCl after 12 h of exposure. Furthermore, the ability of UVC light to inactivate oocysts on smooth surfaces (e.g., laminar flow) was evaluated. To mimic laboratory conditions, oocysts were given on germ carriers. Best results (>99 %) were achieved at an exposure time of 30 min (100.8 mJ/cm2). Keywords Cryptosporidium . Inactivation . UV-C light . Germ carrier Cryptosporidia are important disease agents in humans and animals and may be transmitted from man to man or as zoonotic infection (Lendner et al. 2011). Immunodeficient humans and newborns are especially susceptible and manifest heavy clinical symptoms up to lethal forms of the infection. A main problem in combating the parasite is its resistance against a lot of disinfectants and environmental influences. Inactivation is defined as a reduction of infectious parasite stages. Due to the resistance of the oocysts, a reduction of Cora Delling and Ivette Holzhausen contributed equally to this work. * Matthias Lendner [email protected] 1
Institute of Parasitology, University Leipzig, Leipzig, Germany
infectious parasites by 2 log10 levels is already a good result (Shahiduzzaman et al. 2009). The approved disinfectants are mostly based on chlor-m-cresol which is corrosive and irritating to mucosa. Therefore, cresols are not suitable for the inactivation of oocysts under experimental conditions, e.g. under a laminar flow or in tubings of FACS machines, which makes it difficult to work with this parasites considering that full inactivation has to be achieved following laboratory experimentation. There are many studies dealing with the inactivation of Cryptosporidium parvum oocysts by several substances. Ethanol (Weir et al. 2002), sodium hypochlorite (Fayer 1995; Barbee et al. 1999; Weir et al. 2002; Coulliette et al. 2006) and peroxide (Barbee et al. 1999; Weir et al. 2002; Castro-Hermida et al. 2006) were already tested, but often, exposure times were quiet short and a long course of different exposure times was not undertaken so far. In the present study, such long-term exposure times have been examined. Additionally, the possibility to use UV-C light for inactivating the protozoan was tested. Until now, several studies have shown the effectiveness of UV light to inactivate C. parvum oocysts but most publications deal with the treatment of drinking water (Morita et al. 2002; Clancy et al. 2004) or apple cider (Hanes et al. 2002). While most studies use suspended oocysts, we report on the UV-C light inactivation on smooth surfaces using a germ carrier assay developed to test disinfectants (Dresely et al. 2015). The aim of the study was to find appropriate alternatives regarding inactivation of C. parvum oocysts that can be used under laboratory conditions and are less aggressive than cresols. For both experiments, an in-house strain of C. parvum was used. The maintenance of the parasites was assured through passages in neonatal calves every 3 months. Oocysts were isolated and stored as described earlier (Najdrowski et al. 2007; Dresely et al. 2015). To test alternative disinfectants, 4×105 HCT-8 cells were seeded in 24-well plates with 1 ml growth medium (RPMI 1640, 5 % foetal calf serum (FCS),
864
Parasitol Res (2016) 115:863–866
1 % sodium pyruvate) 24 h before inactivation and infection. C. parvum oocysts were centrifuged for 5 min at 7000g, washed with sterile phosphate-buffered saline (PBS) and resuspended in 1 ml water of standardized hardness for counting in a Neubauer chamber. Oocysts at 3×106 per tube were treated with the test substances. Every test was performed in triplicate. After the treatment, oocysts were centrifuged for 7 min at 15,000g and washed with sterile PBS twice. The oocyst pellet was resuspended in 1 ml growth medium. HCT-8 cells were infected with 2×105 oocysts in the presence of sodium taurocholate (0.2 %), amphotericin B (1 %) and gentamycin (0.5 %). Heat-inactivated oocysts (70 °C for 20 min) were used as positive control, untreated oocysts as negative control and non-infected cells as no template control. After 3 h of excystation (37 °C, 5 % CO2), the cells were washed twice with PBS and provided with new growth medium (supplemented with amphotericin B (1 %) and gentamycin (0.5 %)). After 24 h, the cells were harvested, resuspended in 200 μl PBS and stored at −20 °C until DNA extraction. The DNA extraction was performed with a QIAamp DNA Mini Kit according to the manufacturer’s protocol. The DNA concentration was measured and adjusted to 80 ng/μl. A real-time PCR w a s p er f o r m e d u s i n g t he p r o t o c o l d e s c r i be d b y Shahiduzzaman et al. (2009) with some modifications. The Maxima Probe (Thermo Scientific™) master mix was used, and a standardized amount of 400 ng DNA was applied to each reaction. Each run was performed in technical duplicates. Inactivation using UV-C light was performed in a safety cabinet (HERA SAFE KS 12) with two integrated UV-C ultraviolet lamps (Osram HNS 15W OFR) at both sides emitting light at 254 nm. The UV-C intensity was measured at different points of the working area with a radiation meter (PCE-UV36, PCE Instruments UK Ltd). Inactivation was performed in the centre of the working area. Table 1 Inactivation of C. parvum oocysts by different substances
Treatment
Heat inactivateda Ethanol denatured 100 % ethanol 70 % ethanol 10 % H2O2 3 % H2O2 1.5 % NaOCl 3 % NaOCl 6 % NaOCl
% inactivation±SD 30 min
2h
4h
12 h
24 h
99.96±0.02 97.91±0.22 96.17±0.80 77.80±4.49 97.29±0.14 93.85±3.28 −37.64±6.01 54.32±17.98 88.19±3.43
99.97±0.01 99.21±0.57b 90.41±3.70 96.79±0.55 99.44±0.62c 97.89±0.79 16.17±22.73 95.86±0.81 95.61±1.69
99.94±0.06 98.18±1.54c 91.56±5.51 94.78±1.72 99.68±0.39c 98.31±1.52 89.73±4.02 94.46±1.36 97.84±0.68
99.92±0.06 98.63±0.19 93.89±5.76 84.26±10.40 99.31±0.63c 97.45±1.46 94.53±1.03 99.63±0.32c 99.75±0.20c
99.99±0.02 98.97±0.23 86.24±1.15 88.55±1.47 99.75±0.08 95.76±1.21 97.21±1.48 99.63±0.08 99.91±0.15
n=3 a
To evaluate inactivation by UV-C light, a germ carrier assay as described previously was used (Dresely et al. 2015). Briefly, 1×107 oocysts in 50 μl PBS with 0.03 % bovine serum albumin (BSA) were spread on a 2-cm brushed stainless steel disc and were dried for 30 min in a laminar flow. Subsequently, germ carriers were exposed to UV-C light for 10, 20 and 30 min. To recover the oocysts, germ carriers were transferred into 50-ml Falcon tubes containing 5 ml of distilled water. After 2 min, the Falcon tubes were vortexed for 1 min. The germ carriers were removed and the Falcon tubes were centrifuged at 1000g for 8 min. Infection, harvesting cells and further treatment were done as described above. To test the ability of common chemicals to inactivate C. parvum oocysts, a suspension assay was used. The most promising substances and exposure times were tested twice in triplicate to ensure reproducibility. Heat-inactivated oocysts were used as positive control and exceeded 99.9 % inactivation in all experiments. Highest inactivation (over 99 %) was achieved using 10 % H2O2 at an exposure time of over 2 h as well as 3 and 6 % NaOCl for more than 12 h. All results are summarized in Table 1. Ten percent H2O2 seems to be the best choice as an alternative to cresols regarding inactivation of C. parvum oocysts under experimental conditions, e.g. as a positive control in suspension tests. The results show good agreement with former studies regarding the effectiveness of hydrogen peroxide in cell culture. H2O2 (6 %) was described as an effective disinfectant against C. parvum oocysts (Weir et al. 2002), being able to reduce the oocysts’ infectivity greater than 3 log10 (Barbee et al. 1999). Similarly, Castro-Hermida et al. (2006) tested the commercial disinfectant AGROXIDE II (Laboratoires CEETAL) diluted to 10 % (vol/vol), with the major component being H2O2, in neonatal mice and reported a significantly lower infection (P
