| According to the results of the third survey of the status of key human parasitic diseases in China,it is speculated that the number of people infected with Clonorchis sinensis in China is as high as 15.2 million,and the infection cases account for nearly 50% of the world.Due to the lack of appropriate vaccines and people's dietary habits are difficult to change,drug chemotherapy is still the main means of controlling Clonorchiasis.Studies have shown that praziquantel has a good effect against Clonorchis sinensis,which provides hope for the mass chemotherapy of Clonorchis sinensis.Although praziquantel has been used as the first choice drug for the treatment of Clonorchiasis for more than 30 years due to its high efficiency and low toxicity,there is currently no clear and scientific medication plan and treatment method,and it is currently used clinically to treat Clonorchiasis.The drugs for clonorchiasis infection are mainly racemic(50% each of L-body and D-body)praziquantel.No commercially available levpraziquantel has been seen,nor levpraziquantel has been seen to kill Clonorchis sinensis related research.Studies have shown that L-praziquantel(L-isomer)is the active ingredient of the drug,the dextroisomer has almost no insecticidal effect,and the toxicity to the heart is lower than the dextroisomer.Dextroisomer is considered to be the main cause of side effects and one of the causes of the bitterness and unpleasant smell of drugs.Therefore,it is undoubtedly the most important task to obtain the true effect of levopraziquantel against Clonorchis sinensis and the exact dosage.At the same time,great changes have taken place in the original ecological environment of Clonorchis sinensis natural foci because of the great changes in the way of land use in China in the past few decades.South China,especially the Pearl River Delta,where the Mulberry-Dike-Fish-Pond,streams,and lake blisters that were common in the past have been difficult to see,which led to the difficulty of obtaining Clonorchis sinensis-positive fish for scientific research in the wild.If we completely rely on collecting small freshwater fish from the natural foci of Clonorchis sinensis to obtain the cysticercosis of Clonorchis sinensis,it is not only time-consuming and laborious,but also easily affected by the seasons,which cannot effectively meet the needs of scientific research.In summary,this article on how to establish a scientific,reasonable,economically feasible,easy to operate,not affected by the seasons,and can provide a complete and complete indoor life of Clonorchis sinensis for scientific research.Life cycle has been studied,and on this basis,through laboratory studies to determine the killing effect of L-praziquantel on Clonorchis sinensis,to determine the dose-effect relationship of the drug against Clonorchis sinensis,which is the Provide scientific basis for clinical application.Part ? Construction life cycle of Clonorchis sinensis in laboratory Objective:1.To study the difference in the survival time of Clonorchis sinensis eggs in different solutions at different temperatures,to provide a reference for the preservation of Clonorchis sinensis eggs.2.To study the susceptibility difference and optimal infection conditions of the first intermediate host,second intermediate host and final host of Clonorchis sinensis to provide a reference for the construction of the life cycle in the laboratory of Clonorchis sinensis.Methods:1.Survival time of Clonorchis sinensis eggs in different solutions at 25?Collected the eggs of Clonorchis sinensis with good activity,and observe the eggs of Clonorchis sinensis in deionized water,dechlorinated water,tap water,PBS buffer,0.9% NaCl solution,Alcohol solution,25% glycerol at 25?,1.2% NaCl solution,1.0% sucrose solution and 100% glycerol solution survival time.Trypan blue staining was used to observe the survival of Clonorchis sinensis eggs.2.Survival time of Clonorchis sinensis eggs in dechlorinated water at different temperaturesCollect the eggs of Clonorchis sinensis with good activity and store them in dechlorinated water.Observe the eggs of Clonorchis sinensis at 0?,5?,10?,15?,20?,25?,30? and 35? survival time.Trypan blue staining was used to observe the survival of Clonorchis sinensis eggs.3.Choice of the first intermediate host3.1 First Intermediate Host Susceptibility EvaluationUnder the condition of 25?,according to the snail egg ratio 1:10,1:20,1:30,1:40,1:50,1:60,1:70,1:70,1:90 and 1: 100 respectively Infected snail snail,telephoto snail,Hubei snail,snail umbilical snail,smooth umbilical snail,ring snail and radish snail,100 snails in each group,smash the snail shell after 30 days of infection,check the snail's Infection.3.2 Optimal infection temperatureAccording to the snail-to-oval ratio of 1:60,the snails were infected under the conditions of 20?,22?,24?,26?,28?,30?,32?,34? and 36?,100 snails in each group were infected for 30 days.After breaking the shell,check the infection of the snail under the microscope.4.The choice of the second intermediate host4.1 Second Intermediate Host Susceptibility EvaluationAll the 20 wheat ears used for infection were positive,with a positive rate of 100.00%;only 4 of the 20 bighead carp were positive fish,and the positive rate was 20.00%;only 7 of the 20 Ge's perch carps were positive The positive rate was 35.00% for fish;12 of 20 fish were positive,and the positive rate was 60.00%;all 20 zebrafish were negative.The infection rate of wheat ear fish is much higher than that of the other four kinds of freshwater fish.Statistical analysis shows that the difference in infection rate between wheat ear fish and the other four kinds of freshwater fish is statistically significant(P <0.01).4.2 Optimal infection temperature of wheat ear fishAccording to 50 cercariae per fish,10 fish in each group.Wheat ears were infected at 20?,21?,22?,23?,24?,25?,26?,27?,28?,29?,30?,31?,32?,33?,34? and 35?.All infected fish were killed after 30 days of infection,and cysticercosis was examined by microscopy.5.End host selectionEach experimental mouse was orally gavaged with 50±1 cysticercosis using a gavage needle to infect 30 Balb/c mice,30 CD1 mice,30 C57bl/6 mice,10 Wistar rats and SD,respectively.Ten rats were used.After 40 days of infection,the mice were directly sacrificed by cervical dislocation.The rats were anesthetized with 10% chloral hydrate injection,and then were sacrificed by cervical dislocation after anesthesia.The liver was dissected to check for Clonorchis sinensis in the hepatobiliary duct.Results:1.Survival time of Clonorchis sinensis eggs in different solutions at 25?When stored at 25°C until the 5th day,all eggs of Clonorchis sinensis in 0.9% NaCl solution,Alcohol solution,PBS buffer,25% glycerol,1.2% NaCl solution,1.0% sucrose solution and 100% glycerol died.At this time,the survival rate of eggs in deionized water is 96%,the survival rate of eggs in dechlorinated water is 98%,and the survival rate of eggs in tap water is 67%.There is no difference between the deionized water group and the dechlorinated water group academic significance(P> 0.01),but the differences between the ionized water group and the dechlorinated water group were statistically significant(P <0.01).When stored until the 15 th day,only the eggs in the deionized water group and the dechlorinated water group survived.The survival rate of eggs in deionized water group and dechlorinated water group was 16% and 46%,respectively,and the difference between the two groups was statistically significant(P <0.01).Clonorchis sinensis eggs can survive for up to 35 days in dechlorinated water,and the survival rate of the eggs is 22% when stored until the 35 th day.2.Survival time of Clonorchis sinensis eggs in dechlorinated water at different temperaturesIn the 0°C group,the egg survival rate was only 32% when stored to the 5th day;the egg survival rate had decreased to 0 when stored in the 10 th day.When the 5°C group was stored at the 40 th day,there were still 90% eggs survival rate;when stored to the 60 th day,the egg survival rate is still around 80%.The egg survival rates of the 10°C group,15°C group and 20°C group when stored on the 40 th day were 22%,18% and 12% respectively;the eggs of Clonorchis sinensis stored in dechlorinated water at 5°C,the egg survival rate was still above 80% when stored to the 60 th day.3.Choice of the first intermediate host3.1 First Intermediate Host Susceptibility EvaluationAmong the seven kinds of freshwater snails selected,only the biopsy snails and snails can infect Clonorchis sinensis and can be used as the first intermediate host of the life cycle of Clonorchis sinensis.Among the 10 experimental groups infected with Macrobrachium snail,the infection rate of the 1:60 group and the 1:90 group was the highest,both 22%.There were no statistically significant differences in infection rates between the 1:50 group,1:60 group,1:70 group,1:80 group,1:90 group,and 1: 100 group(P>0.01),but the difference in mortality between the: 50 group,1:60 group and 1:70 group,1:80 group,1:90 group and 1: 100 group was statistically significant(P<0.01),but from 1:70 group.Initially,the mortality rate of P.snails increased with the increase of infection.The maximum infection rate of S.hornworm is about 18.5%,but from the 1:40 group,the snail horned snail died in large numbers as the amount of infection increased.3.2 Optimal infection temperatureAccording to the snail-to-oval ratio of 1:60,the infection rate of the snail snail is increased.When the temperature is increased from 20°C to 28°C,the infection rate of the snail snail gradually increases,and the infection rate can reach up to 22.00% at this time;At 36°C,the infection rate of Marsh snails gradually decreased to 6.00%.Statistical analysis showed that the differences between the 24°C,26°C and 28°C infection groups were not statistically significant(P>0.01),but the differences between the 24°C,26°C and 28°C infection groups and other groups were statistically significant Academic significance(P<0.01).4.The choice of the second intermediate host4.1 Second Intermediate Host Susceptibility EvaluationAll the 20 wheat ears used for infection were positive,with a positive rate of 100.00%;only 4 of the 20 bighead carp were positive fish,and the positive rate was 20.00%;only 7 of the 20 Ge's perch carps were positive.The positive rate was 35.00% for fish;12 of 20 fish were positive,and the positive rate was 60.00%;all 20 zebrafish were negative.The infection rate of wheat ear fish is much higher than that of the other four kinds of freshwater fish.Statistical analysis shows that the difference in infection rate between wheat ear fish and the other four kinds of freshwater fish is statistically significant(P <0.01).4.2 Optimal infection temperature of wheat ear fishWhen the temperature increased from 20°C to 27°C,the infection rate and the rate of cystic larvae gradually increased;when the temperature was 27°C,an average of 40.33±4.22 cystic larvae could be collected in each wheat ear fish.However,when the temperature continued to rise to 35°C,the infection degree and the rate of cystic larvae gradually decreased.When the infection temperature was 35°C,only 5.68±3.31 cystic larvae could be collected in each wheat ear.After statistical analysis,the differences between the 27°C group and the 28°C group were not statistically significant(P> 0.01),but the differences between the 27°C group and 28°C and other groups were statistically significant(P < 0.01).5.End host selectionOf the five strains of experimental mice used in this study,only SD rats,Wistar rats and Balb/c mice were successfully infected with Clonorchis sinensis.The positive rate and adult acquisition rate of SD rat infection group and Wistar rat infection group were 100.00%,and the detection rates were 50.80% and 49.80%,respectively.The positive rate of the Balb/c mice infection group was 46.67%,and a total of 96 Clonorchis sinensis were detected,of which 10 were adults.The adult acquisition rate and detection rate were 10.42% and 19.20% respectively.The difference between the SD rat infection group and the Wistar rat infection group was not statistically significant(P>0.01),but the difference between the SD rat infection group and the Wistar rat infection group and the Balb/c mouse infection group had Statistical significance(P<0.01).Part ? Therapeutic effect of L-praziquantel on Clonorchis sinensis infection Objective:1.To study the killing effect of L-praziquantel on Clonorchis sinensis at different ages to determine the dose-effect relationship of the drug against Clonorchis sinensis,so as to provide a scientific basis for the clinical application of the drug.2.To study the insecticidal effect of L-praziquantel,D-praziquantel and racemic praziquantel on 5 weeks old Clonorchis sinensis adults to determine the killing effect of levpraziquantel when killing insects with the same dose of three drugs whether the insect effect is optimal.Methods:1.Experimental animal modeling210 SD rats were orally gavaged with a gavage needle,each rat was gavaged with 50±1 cysticercosis,and 7 rats/cage were housed in a laboratory animal room(SPF)with 12 h light cycle and constant temperature control,without restricting drinking water.2.Laboratory animalsIn this study,a single intragastric administration method was used.The experimental group was administered by oral gavage at 0.1mL/10 g per rat body weight.The control group was given 2.5% EL by oral gavage according to the body weight of each rat.Results:Lepraziquantel has a strong killing effect on clonorchiasis sinensis at different ages in rats.Among them,the deworming rate of 37.5mg/kg levaziquantel on 1 week old Clonorchis sinensis larvae was as high as 92.30%;the 75mg/kg levaziquantel on 2 week old Clonorchis sinensis larvae was reduced up to 95.00%;75mg/kg levaziquantel has a deworming rate of 3 weeks old adults of Clostridium sinensis and 83.87%;75mg/kg levaziquantel has a deworming rate of 4 weeks old adults of Clonorchis sinensis up to 74.52%,but when the drug concentration is increased to 150mg/kg,the worm reduction rate is as high as 97.52%;150mg/kg levaziquantel has a worm reduction rate of 93.17% against clostridium sinensis adults,even if the dose is reduced to 75mg/kg still has 58.39% insect reduction rate. |
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