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 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), 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 was performed using the protocol described by 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.

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 < 0.01) than in control litters. The concentration of 3 % of H2O2 was obviously too low to reach the intended inactivation rate. In thoughts of practicability and costs, denatured ethanol (100 % ethanol, 1 % methyl ethyl ketone (MEK); Brüggemann Alcohol Heilbronn), 70 % ethanol (commonly used as surface disinfectants in many laboratories) and 100 % ethanol were tested. Incubation with 70 and 100 % ethanol resulted in high variations of inactivation. Weir et al. (2002) observed no reduced infectivity of C. parvum oocysts in cell culture after 33 min of exposure to 70 % ethanol. The presented data demonstrate that longer exposure times enhance the efficacy of 70 % or even 100 % ethanol. Nevertheless, the inactivation rate did not exceed 98 %. Dawson et al. (2004) tested different treatments including 9 and 40 % ethanol to determine their effects on survival of C. parvum and Cryptosporidium hominis oocysts using sporozoite ratio and infection of MRC-5 cells. The reduction of cell culture infectivity was only 77 % after treatment with 9 % ethanol for 7 days and 72 % after treatment with 40 % ethanol for 8 days using C. hominis oocysts. These results show good agreement with those of the present study. Interestingly, denatured ethanol in comparison to non-denatured ethanol showed a higher inactivation. That difference seems to be related to the composition of denatured ethanol. In this study, the used denatured ethanol was composed of 99 % ethanol and 1 % MEK. MEK is often used as an admixture to produce denatured ethanol but also as a solvent or for sterilization of medical instruments. So probably the combination of both components leads to a better inactivation effect as compared to pure ethanol. Although the results of former studies suggest a minor efficacy of NaOCl, we tested three concentrations to check the inactivation efficacy after longer exposure times. After 30 min exposure to 1.5 % NaOCl, an activation of oocysts was reported by Coulliette et al. (2006) probably due to the destruction of the glycocalyx and therefore better accessibility of bile salts to the oocyst. Accordingly, we found an increased infectivity at the lowest concentration (1.5 %) and shortest incubation time (30 min) but prolonged incubation resulted in increasing inactivation reaching over 99 % when incubated with 3 or 6 % NaOCl for at least 12 h. Fayer (1995) tested the effect of sodium hypochlorite (5.25, 2.63 and 1.31 %) in a neonatal mouse model and reported lower histology scores. However, parasite stages were still identified. Barbee and colleagues showed a decrease in infectivity of C. parvum by <1 log after exposure to Clorox (NaOCl, 5.25 %) (Barbee et al. 1999), and Weir et al. (2002) described a poor effect of sodium hypochlorite (6 %) up to an exposure time of 33 min. The present study confirms that although sodium hypochlorite activates C. parvum oocysts at low concentrations over short incubation periods, it can be used as disinfectant for laboratory tubings and containers when exposure times are increased.

Table 1 Inactivation of C. parvum oocysts by different substances
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Next, it was of interest to know if spilled and dried oocysts on a smooth surface (e.g. the working area of a safety cabinet) can be inactivated by UV irradiation. To date, several studies have shown the effectiveness of UV irradiation to inactivate C. parvum (Belosevic et al. 2001; Hanes et al. 2002; Morita et al. 2002; Clancy et al. 2004) or C. hominis (Johnson et al. 2005) oocysts in suspension. We tested three different exposure times (10, 20 and 30 min) at a UV intensity of 0.056 mW/cm2 on oocysts sticking to a germ carrier.

First, the UV-C intensity at different positions of the safety cabinet was measured showing an uneven distribution of the UV-C intensity (Fig. 1). Inactivation of the oocysts was assessed in the centre of the working area where most actions take place. The results of the exposure of C. parvum oocysts to UV irradiation are shown in Fig. 2. An inactivation of over 99 % was achieved at a dose of 100 mJ/cm2 (30 min exposure time). Exposure times of 10 min (33.6 mJ/cm2) and 20 min (67. 2 mJ/cm2) led to an inactivation of 97.17 % (±1.72 %) and 98.99 % (±0.48 %). The results confirmed the detrimental effects of UV light on oocysts of C. parvum. Whereas in other studies the oocysts were in suspension while being exposed to UV light, in this study, the oocysts stuck to a germ carrier. A 3.2 log10 inactivation at a UV dose of 2 mJ/cm2 and 4.1 log10 inactivation at 4 mJ/cm2 were reported for the Iowa strain (Clancy et al. 2004). Interestingly, Morita et al. (2002) reported that even a low UV dose of 1.92 mJ/cm2 was enough to reduce infectivity in mice by 4 log10 levels whereas 230 mJ/cm2 was necessary to reduce excystation by 2 log10 levels. Thus, the long-term effects of UV irradiation were more pronounced in an infection model than in the excystation model. This might explain why in the present study relatively high UV doses were necessary. The ability of sporozoites to invade and proliferate in cells was assessed 24 h post infection when the life cycle is not completed. Hence, long-term effects are not detected by this approach. The absence of light or dark repair of DNA damages in C. parvum oocysts after treatment with low-pressure UV light was reported in two independent studies, indicating that the DNA damage is not reversible (Shin et al. 2001; Rochelle et al. 2002). Altogether, this report proves that UV inactivation is an effective and also persistent method to inactivate C. parvum oocysts.

Fig. 1
figure 1

Measured UV-C intensity within the safety cabinet; intensity in microwatts per square centimetre; white circles UV source (Osram HNS 15W OFR), blue circles measuring points with detected UV-C intensity, red circle inactivation assay, grey boxes area of ventilation

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Fig. 2
figure 2

Inactivation of C. parvum oocysts by UV irradiation; comparison of UV-C-irradiated C. parvum and heat-inactivated (h.i.) oocysts (70 °C, 20 min)

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In summary, we demonstrated the effectiveness of 3 % NaOCl for 12 h or, alternatively, the use of 10 % H2O2 for 2 h to inactivate C. parvum oocysts and moreover that UV-C irradiation (>100 mJ/cm2) is an appropriate method to inactivate C. parvum oocysts on smooth surfaces.