Cryptosporidium spp. ookistlerinin gama ışınlama ile inaktivasyonu
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Date
2017
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Publisher
Türkiye Atom Enerjisi Kurumu
Abstract
Cryptosporidium türleri, sularla bulaştığı bilinen ve özellikle bağışıklık sistemi baskılanmış bireylerde şiddetli ishal oluşturan gastrointestinal, hücre içi protozoon parazitlerdir. Bu organizmanın zor çevre koşullarına ve çeşitli dezenfektanlara karşı dirençli olduğu, gama ışınlarına karşı yüksek direnç gösterdiği bilinmektedir. Bu parazitin çevre örmeklerinde inaktivasyonunu sağlayan değerlerin bilinmesi halk sağlığı açısından büyük önem taşımaktadır.
Bu çalışmada gama ışınlamanın Cryptosporidium ookistlerinin canlılığı ve enfektivitesi üzerindeki etkisini belirlemek üzere başlıca iki yöntem kullanılmıştır:
- Ookistlerin canlılığını belirlemek amacıyla kullanılan vital boyalar : 4’,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI) ve SYTO-59.
- Ookistlerin açılım yeteneğini belirlemek amacıyla uygulanan in vitro ekskistasyon yöntemi.
Araştırmada kullanılan ookistler doğal yolla enfekte olmuş dana dışkılarından elde edildi. Ookistlerin konsantrasyon ve saflandırma işlemleri için şeker flotasyon ve sezyum klorür tabakalandırma yöntemleri kullanıldı. Su örnekleri suya 106 oocysts/ml ookist ekilerek hazırlandı. Örnekler 60Co kaynağında 1, 5, 10, 15 and 20 kGy dozlarda ışınlandılar. Işınlanan örnekler 4°C’de 10 gün bekletildikten sonra ışınlama sonrası zamana bağlı etkiler açısından tekrar incelendiler.
Vital boyalarla yapılan testler 15 kGy dozda ışınlanan örneklerde ookistlerin canlı olmadığını gösterdi. Çalışma ekskistasyon ve canlılık oranlarının gama ışınlanma dozu ile ters orantılı olduğunu gösterdi. Işınlanmamış kontrol örneklerde açılım oranı %90 civarında, canlılık oranı DAPI/PI ile %84.03, SYTO-59 ile %90.48 olarak saptandı. 15 kGy dozda açılım gösteren ookistlerin 10 gün sonra bu yeteneklerini kaybettikleri gözlendi. Bu durum gama ışınlamaya tabi tutulan ookistlerin açılabildiklerini fakat Pl ve SYTO- 59 boyalara karşı geçirgenlik kazandıkları için enfektif olamayacaklarını ortaya koydu.
Aynı testler Ankara Merkezi Atıksu Arıtma Tesisi’nden alınan arıtılmış su örneklerinde de tekrarlandı. 10 kGy dozda boş ve/veya yarı açılmış ookistlere ve serbest sporozoitlere rastlanmadı. Işınlanmamış örneklerde ekskistasyon oranı %92 olurken 10 kGy dozda açılım görülmedi. Kontrol örneklerinde canlılık yüksek bulundu ( DAPI/PI ile %72.8, SYTO-59 ile %85.2). Canlılığın 5 kGy dozda azaldığı ve 10 kGy dozda ookistlerin tamamen öldükleri görüldü (Pl ve SYTO-59 pozitif).
Sonuç olarak, 15 kGy dozda İyonlaştırıcı radyasyonun Cryptosporidium ookistlerinin açılım yeteneğini engellediği ve canlı sporozoit taşıma oranını azalttığı belirlenmiştir. Su ve çevre örneklerinin dezenfeksiyonu amacıyla uygulanan yöntemlerin ookistler üzerindeki etkisinin belirlenmesinde vital boya ve in vitro ekskistasyon testlerinin güvenle kullanılabileceği görülmüştür
Cryptosporidium species are well-known waterborne intracellular protozoan parasites that cause severe diarrheal illness especially in immunocompromised individuals. The organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits high resistance to gamma irradiation. The ability to determine inactivation rates of Cryptosporidium oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite. The present study was undertaken to determine the effects of gamma irradiation on viability and infectivity of Cryptosporidium oocysts by using dye permeability assay, which tests the differential uptake of the fluorochromes 4’,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI) and SYTO-59 by the oocysts in vitro excystation assay, which tests oocysts’ ability to excyst. Wild-type oocysts were obtained from naturally infected calf feces and concentrated and purified by sucrose flotation and cesium chloride gradient techniques. Spiked samples (106 oocysts/ml) were prepared for the examination of water samples. Samples were gamma irradiated at 60Co source at various doses (1, 5, 10, 15 and 20 kGy) and were incubated at 4°C for 10 days for the further examination of time dependent post irradiation effects. Oocysts that received 15 kGy gamma irradiation dose were not viable as indicated by the dye permeability assay. The study indicated that the gamma irradiation doses and excystation and viability rates were inversely proportional. Non irradiated (control) oocysts excysted at rate of about 90%, and the viability rates of non irradiated oocysts were 84.03% by DAPI/PI and 90.48% by SYTO-59. At the dose of 15 kGy the irradiated oocysts were still capable to excyst, however, after ten days they had completely lost the ability to excyst. These results indicated that gamma irradiated oocysts may still be capable of excystation however, as they were permeable to PI and SYTO-59 they must not have been infective. The treated wastewater samples collected from the Ankara Central Municipal Wastewater Treatment Plant were examined similarly. At the dose of 10 kGy neither partly excysted and/or empty oocysts nor free sporozoites was seen. Untreated control oocysts maintained 92% excystation rates, but oocysts receiving 10 kGy dose of irradiation were completely incapable of excystation. While the control oocysts were highly viable (72.8% by DAPI/PI and 85.2% by SYTO-59), the viability is reduced at the dose of 5 kGy, and the oocysts irradiated with 10 kGy were mainly nonviable (PI or SYTO- 59 positive). In conclusion, 15 kGy dose of ionizing irradiation prevents the ability of Cryptosporidium oocysts to excyst and renders the contained sporozoites nonviable. Vital dye based assays and in vitro excystation techniques are good indicators of viability to examine the effects of disinfection processes in water and environmental samples.
Cryptosporidium species are well-known waterborne intracellular protozoan parasites that cause severe diarrheal illness especially in immunocompromised individuals. The organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits high resistance to gamma irradiation. The ability to determine inactivation rates of Cryptosporidium oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite. The present study was undertaken to determine the effects of gamma irradiation on viability and infectivity of Cryptosporidium oocysts by using dye permeability assay, which tests the differential uptake of the fluorochromes 4’,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI) and SYTO-59 by the oocysts in vitro excystation assay, which tests oocysts’ ability to excyst. Wild-type oocysts were obtained from naturally infected calf feces and concentrated and purified by sucrose flotation and cesium chloride gradient techniques. Spiked samples (106 oocysts/ml) were prepared for the examination of water samples. Samples were gamma irradiated at 60Co source at various doses (1, 5, 10, 15 and 20 kGy) and were incubated at 4°C for 10 days for the further examination of time dependent post irradiation effects. Oocysts that received 15 kGy gamma irradiation dose were not viable as indicated by the dye permeability assay. The study indicated that the gamma irradiation doses and excystation and viability rates were inversely proportional. Non irradiated (control) oocysts excysted at rate of about 90%, and the viability rates of non irradiated oocysts were 84.03% by DAPI/PI and 90.48% by SYTO-59. At the dose of 15 kGy the irradiated oocysts were still capable to excyst, however, after ten days they had completely lost the ability to excyst. These results indicated that gamma irradiated oocysts may still be capable of excystation however, as they were permeable to PI and SYTO-59 they must not have been infective. The treated wastewater samples collected from the Ankara Central Municipal Wastewater Treatment Plant were examined similarly. At the dose of 10 kGy neither partly excysted and/or empty oocysts nor free sporozoites was seen. Untreated control oocysts maintained 92% excystation rates, but oocysts receiving 10 kGy dose of irradiation were completely incapable of excystation. While the control oocysts were highly viable (72.8% by DAPI/PI and 85.2% by SYTO-59), the viability is reduced at the dose of 5 kGy, and the oocysts irradiated with 10 kGy were mainly nonviable (PI or SYTO- 59 positive). In conclusion, 15 kGy dose of ionizing irradiation prevents the ability of Cryptosporidium oocysts to excyst and renders the contained sporozoites nonviable. Vital dye based assays and in vitro excystation techniques are good indicators of viability to examine the effects of disinfection processes in water and environmental samples.
Description
TENMAK D.N.. 10554
Keywords
Gamma rays, Gama ışınları, Gamma irradiation, Gama ışınlaması, Invitro excystation, In vitro ekskistasyon, DAPI, PI, SYTO-59
Citation
Emre, Z. ...[ve arkadaşları]. (2017). Cryptosporidium spp. ookistlerinin gama ışınlama ile inaktivasyonu. Ankara : Türkiye Atom Enerjisi Kurumu.