Effects of Gamma Irradiation on the Survival of Encephalitozoon intestinalls Spores
Encephalitozoon intestinalis is a fecal and urine borne pathogen of humans. The specific routes of transmission have not been well documented, but like other fecal borne pathogens the infectious stage can be expected to be transmitted by direct contact or by ingestion of contaminated food and water. Irradiation of food products can reduce or prevent infection with microbial pathogens. No data are presently available indicating the levels of irradiation that may be useful for reducing or eliminating infectivity of any microsporidian species.The present study was conducted to determine the effects of graded doses of gamma irradiation on infectivity of E. intestinalis spores.
MATERIALS AND METHODS
Encephalitozoon intestinalis spores (ATCC no. 50604) were obtained from the American Type Culture Collection (Manassas, VA). Spores were propagated in MDBK monolayer cultures in T-75 flasks in DMEM medium with 10% fetal calf serum and non-essential amino acids. Spores were harvested from cell cultures and counted with the aid of a hemocytometer. To determine the optimum number of spores to use as inoculum in a bioassay, a titration was conducted in which 5000 to 50,000 spores were inoculated into each of 2 wells of an 8 well Lab-Tek chamber slide (Nalge Nunc Int'l, Naperville, IL) containing a monolayer of MDBK cells. After 4 days of incubation at 35 C in a 5% CO2 atmosphere incubator, culture medium was removed and cultures were fixed in situ with 100% methanol. After drying cells were stained with hot Gram-Chromotrope stain [1]. The number of intracellular clusters of developing microsporidia in each well were enumerated by brightfield microscopy.
For exposure to gamma irradiation, a total of 9 x 106 spores were suspended in 800 μ of DMEM and placed in a 15 ml centrifuge tube. The tube was exposed to gamma irradiation in a 137Cs Gammator M irradiator at 20 C. Dosage was cumulative over time. Upon exposure to 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 1.0 kGy respectively, 150,000 spores were removed at each dosage level. To determine infectivity of the spores at each dosage level, 50,000 of these spores were inoculated into each of 3 wells of an 8 well chamber slide, each well containing a monolayer of MDBK cells. After 4 days incubation, fixation, staining, and enumeration of developing microsporidia were conducted as described above.
RESULTS AND DISCUSSION
The optimum number of spores for inoculation into monolayer well cultures of MDBK cells was found to be 50,000. This number was used to test infectivity of spores exposed at all gamma irradiation levels.
Distinct reductions in the number of clusters of microsporidia were observed as the dose of gamma irradiation increased beyond 0, 0.1 and 0.2 kGy (Table 1). Between 0 and 0.1 kGy a statistically significant increase was observed, possibly suggesting that slight irradiation might promote infection or germination of spores.
| no. of intracellular clusters per well | ||||||
|---|---|---|---|---|---|---|
| kGy | Well 1 | Well 2 | Well 3 | Mean | SD | P value |
| 0 | 129 | 115 | ND | 122 | 9.9 | |
| 0.1 | 134 | 142 | 150 | 142 | 8.0 | * |
| 0.2 | 130 | 122 | 111 | 121 | 9.5 | NS |
| 0.25 | 79 | 66 | 69 | 71.3 | 6.8 | *** |
| 0.3 | 71 | 70 | 75 | 72 | 2.6 | *** |
| 0.4 | 46 | 38 | 29 | 37.7 | 8.5 | *** |
| 0.5 | 11 | 18 | 16 | 15 | 3.6 | *** |
| 0.6 | 0 | 1 | 0 | 0.3 | 0.6 | *** |
| 0.7 | 1 | 0 | 0 | 0.3 | 0.6 | *** |
| 0.8 | 1 | 1 | 0 | 0.7 | 0.6 | *** |
| 1.0 | 0 | 1 | 0 | 0.3 | 0.6 | *** |
- ND = Not Done SD = Standard Deviation P value based on Tukey-Kramer Multiple Comparisons Test; none vs all others; *= P<0.05, ***= P<0.001
A mean of 129 clusters resulting from an inoculum of 50,000 spores indicates that only one spore out of every 388 that were inoculated underwent development in cells. Despite this finding that suggests a relatively low level of infectivity to begin with, at exposure levels of 0.6 kGy and higher the mean number of clusters was 0.4 per well, representing only 0.3% of the clusters found after exposure to 0,0.1 and 0.2 kGy, a decrease of 99.7%.
This is the first report of an attempt to determine the effect of gamma irradiation on infectivity of any species of microsporidian parasite.
Coccidia are another group of protozoan parasites with an encysted exogenous stage (the oocyst) that contaminates the environment and initiates infections in other hosts. Unsporulated and sporulated oocysts of Toxoplasma gondii were irradiated to determine the dose to sterilize contaminated fruit [2]. Unsporulated oocysts irradiated at ≥0.4 to 0.8 kGy sporulated but did not infect mice. Sporulated oocysts irradiated at≥0.4 kGy did not infect mice. A dose of 0.5 kGy was considered effective for “killing” oocysts on fruits and vegetables. Results from the present study indicate that even at doses 2X greater than that required to kill T. gondii, a few E. intestinalis spores remained infect-ious. Another coccidian parasite, Eimeria tenella, had 77–94% reduced development in cell culture after exposure to 0.05–0.2 kGy [3] and exposure to 0.05 kGy to render oocysts noninfectious for chicks [4].
A wide range of irradiation doses are required to render food safe from microbial pathogens, depending primarily on the species of organism associated with the food product and the temperature at the time of irradiation [5], For example, radiation doses of 2–7 kGy can effectively eliminate potentially pathogenic non-sporeforming bacteria such as Salmonella, Staphylococcus, Campylobacter, Lisleria, and Escherichia coli O157:H7 [6]. Irradiation doses of 0.15–0.7 kGy under specific conditions appears feasible for controlling many foodbome parasites [6].
The present study has demonstrated that infectivity of the microsporidium E. intestinalis is greatly reduced but not completely eliminated at irradiation doses of 0.6–1.0 kGy.
ACKNOWLEDGEMENTS
The authors thank Elizabeth Didier for suggestions and guidance.
