Inactivation Effects And Mechanisms Of Cryptosporidium Parvum Oocysts In Water By Chlorine And Ozone

Cryptosporidium is the most common and harmful parasite which can infectsintestinal epithelia of humans and animals, causing diarrhea through drinking watercontaminated by Cryptosporidium. The water is the key route of transmission. Watercontaminated with Cryptosporidium tends to result in the outbreak ofCryptosporidiosis. Cryptosporidiosis is known to one of the most common diarrheadiseases in the world. Currently, no effective drugs can cure cryptosporidiosis.Oocysts are very small. Cryptosporidium oocysts have a certain resistance not only tothe various environmental factors, but also to conventional water treatment process.Conventional chlorine disinfection almost cannot inactivate Cryptosporidium oocysts,which has posed a serious threat to drinking water safety. Cryptosporidium had beenregarded as microbiology standards of water quality in developed countries.Cryptosporidium was very important and urgent for control measures research in thewater.In this study fluorogenic vital-dye assays was regarded as the tools of assessingthe viability of Cryptosporidium parvum oocysts. We studied the inactivationprinciple and effect of the two disinfectants, also initially explored the inactivatingmechanism of Cryptosporidium parvum oocysts in water with chlorine and ozone.The inactivated measures of Cryptosporidium in the water provided a theoreticalreference. The main research content and results were as follows:1. Amplification and recovery Cryptosporidium parvum oocysts were amplifiedthrough BALB/c mice. We developed and evaluated four methods for isolatingCryptosporidium parvum oocysts from the feces of infected mices that used thesaturated sodium chloride, zinc sulfate saturation, discontinuous sucrose densitygradient centrifugation and saturated zinc sulfate - discontinuous sucrose densitygradient centrifugation, respectively. And then we compared four methods inseparating Cryptosporidium oocysts from feces. The result show that 0.15mgdexamethasone (DEX) per mice for seven day can suppressed the immunosystembefore the mices were inoculated intragastrically with Cryptosporidium parvumoocysts. Then the mices were immunosuppressed with 20 mg/L DEX in drinking water. The mice is able to infection successfully, we get experimental repeatabilityand model stability. Among the purification methods, discontinuous sucrose densitygradient centrifugation purification did not affect the activity of oocysts. Purifiedoocysts were higher purity than another three methods, and gave a higher recovery.2. Detection and identification of oocysts Cryptosporidium oocysts weredetected with modified acid-fast stain, general PCR and nested-PCR, respectively.The results showed that the capacity of modified acid-fast staining was limited, whichcould be used only as a preliminary screening method in fecal. Detection specificityand sensitivity of general PCR are better. By the improvement of methods of brokenoocysts, the ultr-asonic fragmentation group raised more an order of magnitude thanfreeze-thaw testing group. The modified methods could detect 2 oocysts per milliliter,the sensitivity of nested-PCR was even better than that of the general PCR, which candetecte less than 1 oocysts per mL.3. Evaluation of disinfection effect by ozone and chlorine The disinfectionconditions, such as the different doses of disinfectants, contacting time, watertemperature, pH value were studied. The results showed the effect and speed ofinactivation on Cryptosporidium parvum oocysts in water with Ozone is better thanthat of chlorine. 99.99% of the oocysts were inactivated when exposed for 3 min with3 mg/Lof ozone, while 3mg / Lof chlorine exposed for 120min with inactivation ratereached only 37.43%. With the increasing of water temperature (4℃, 20℃, 30℃),inactivation rates by ozone were decreasing. And lower temperature was conducive toozone disinfection. With the increase of pH value (6.0, 7.0, 8.0) , the inactivation ratebecame lower byozone, and low pH value had good benefit for ozone.4. Disinfection mechanisms of ozone and chlorine Cryptosporidiumparvum oocysts were inactivated with different concentrations of ozone and chlorineand disinfection time, respectively, disinfection time of low concentration ozone wasfive minute, that of high concentrations ozone was the same ten minute as lowconcentration chlorine, that of high concentration chlorine was 120 minute. Themechanism research of the two disinfectants include three aspects, observing theultra-structural changes by electron microscopy, detection of enzyme activity change,and analying nucleic acid changes withAFLP.Ultra-structure study: surface of oocyst in control group is smooth, circular andspherical by SEM. With the increase of the disinfectant concentration, oocysts peplosbegan to fold. In the high concentration of ozone and chlorine groups, serious fold appeared in the surface of some oocysts. In addition, some oocysts lost their circularstructure, even completely destructed, oocyst sporozoites had overflow, and oocystbecame an empty shell. From the TEM, the peplos of the normal oocyst had anintegrity structure, and content distribution was uniform. With the increases ofexposing time by ozone and chlorine, the surface of oocysts membrane began toshrink, the membrane structure was damaged. Then contents of oocysts began to leakout. Some oocysts were damaged completely and became shell.Enzyme activity changes: MDA appeared to increase, and T-AOC became low,when the concentration of ozone and chlorine increased. Compared with blank controlgroup, p<0.05. The results showed 3mg/L ozone or 60mg/L chlorine damagedanti-oxidative system of oocysts. With regard to energy-related enzymes, the vitalityof ATP in high concentration groups decreased more observely than that of controlgroup. However, there were not significantly different in the aspect of LDH vitalityamong blank control group, high and low concentrations treatment group.Analysed nucleic acid changes: In this study, except the research of ultrastructureand enzyme activity, amplified length polymorphism (AFLP) analysed whetherdisinfectants had a role on the nucleic acid. AFLP had high accuracy, repeatability,and reliability characteristics. The technical steps included DNAextraction, digestion,ligation, pre-amplification, selective amplification, PAGE and silver staining. If thesame fragment was not damaged by chlorine and ozone, treatment group anduntreated group by PAGE had a similar fragment in specific position. Otherwise,PAGE appeared to the discrepancy fragments. In the experimental genomic wasdigested doubly by EcoRI and MseI. We desinged 20 pairs of primers foramplification of each group, and as much as possible understanded the role of theentire genome through chlorine and ozone, the treatment group and untreated groupwere amplified by the same primers, the PAGE showed that the different fragmentsappeared between treated group and untreated group. According to the frequency offragments, different fragments can be divided into 4 categories, this manifested thatchlorine and ozone had an impact on the genetic structure in the treatment group.The results of this study were analysed comprehensively, we had reached thefollowing conclusions:1. Ozone inactivation effect is better for oocysts than that of chlorine. Inactivationeffect of Cryptosporidium oocysts with chlorine was poor. Chlorine disinfectionconcentration of drinking water in our country does not exceed 4mg /L, our experimental results showed that inactivation effect by3mg/Lchlorine was poor,3mg/L chlorine was unfeasible in practical applications. Ozone inactivation waseffective for oocysts, concentration of ozone water disinfection in our countrydoes not exceed 0.3mg/L. 0.3mg/L ozone could achieve more than 99% for60min from our experiment results. Because half-life of ozone was short, it issuggested that Cryptosporidium parvum oocysts was disinfected byozone firstly,then oocysts were inactived by chlorine again.2. Injury of oocysts capsule structure was consistent to antioxidant enzyme activitychanges in the system, This was mainly caused by the oxidation of chlorine andozone,Oxidation was the primary mechanism for inactivation of oocysts.Chlorine and ozone can cause genomic DNAdamage of oocyst, whether there areother mechanisms on genome damage, further experiments needed be explored.Among capsular structure, the enzyme activity and the genome, the sequence ofdestruction by chlorine and ozone needed studyfurtherly.3. AFLP technology can analyze genome injury, it provided a basis for technologyof disinfection mechanism4. Animal infection model of Cryptosporidium parvum was established successfully.Discontinuous sucrose density gradient centrifugation is an ideal method of thepurification oocysts from mouse feces.5. The specificityand sensitivityof PCR detection was better. Nested-PCR detectionsensitivityis higher than the common PCR.Activitystaining method can be usedfor activityidentification of oocysts and evaluation of disinfection effect.