| Objective:This study consists of two parts:the first part is to study the pathogenicity of Toxo DB#9strain of T.gondii from the aspects of immunology,molecular biology and pathology and to understand the virulence characteristics of Toxo DB#9 strain of T.gondii.The virulence characteristics provide experimental data for further study on the pathogenic mechanism of T.gondii infection host.The second part:investigate the infection of T.gondii in ethnic minority areas of Mojiang County and analyze its risk factors,provide a scientific basis for eugenetics.Method:Part ?:(The virulencestudy of T.gondii strains in Dali of Yunnan province)Toxo DB#9T.gondii tachyzoites were preserved in the laboratory.200 mice which aged 6-8 weekswere randomly divided into10 groups(eight ToxoDB#9 groups,one control group and one RH strain group),20 in each,sexual ratio was 1:1.Inoculation by intraperitoneal injection,eight ToxoDB#9 groups include:10~0,10~1,10~2,10~3,10~4,10~5,10~6,10~7;RH strain group inoculated10~3,and the control group injection Physiological saline,each component is kept in cages,and drinking water is taken in the same environment.After the infection treatment,the diet,activity,mental state and morbidity of the mice were observed every day.The body weight of the mice was weighed every other day,the body weight changes were recorded,the time of death of the mice was recorded.On the 5th,10th,20th,30th,and 45th day after infection,one male and one female were randomly selected from each group and the eyeballs were taken for blood collection,the supernatant was centrifuged,and the mouse serum T.gondii IgG antibody was detected by enzyme-linked immunosorbent assay(ELISA),and the spleen tissue was taken for PCR to detect the characteristic B1 gene of T.gondii to determine the infection and death of the mice in order that countthe infection rate and mortality of the mice.The definitions of 10~2,10~4,and 10~6 groups were set as low,medium,and high dose groups.On the3rd,7th,15th,and 45th day after infection,1 male and female mice were randomly selected from the low,middle,and high dose groups,the control group,and the RH strain group.The eyeballs were taken from the eyeball and the supernatant was separated.Serum cytokines IL-2,IL-12 and IFN-?were detected by ELISA.Under sterile conditions,each tissue was taken out and immersed in formalin fixative.The next day,conventional paraffin sections were stained with HE to examine pathological changes.Part ?:The original data of the sero-epidemiology survey of T.gondii in Mojiang County,Yunnan Province comes from 311 newborns and corresponding maternal births in the Obstetrics and Gynecology Department of Mojiang County People's Hospital from October2016 to April 2017.In this study,the serum T.gondii IgG antibody was detected by ELISA,and the differences among different ethnic groups,fetal sex,maternal age,pregnancy history and the positive rate of T.gondii serum antibody were analyzed by the X~2 test method.Result:1.1 In the first part of the study,the mice after 24 hours of intraperitoneal injection in the control group of RH strains were apathetic and the activity was slightly decreased.The mental status of the mice in the to 10~5 ToxoDB#9 concentration group decreased slightly,the activity was slightly reduced,and the drinking water was normal.The mice in the?10~4concentration group and the blank group had normal feeding,drinking water and activities.After 48h,the control group of RH strains had poor mental condition,and the intake of drinking water was reduced.The conjunctiva of one mouse showed redness and swelling.The mental state of mice in the?10~3 ToxoDB#9 concentration group decreased slightly.Hi lying,slightly bowed,eating and drinking water normal;?10~2 concentration group and blank group mice were normal,eating,drinking,and activities.After 72 hours of injection,the mice in the control group of RH strain and the?10~3 dose group were depressed and happy,and the amount of drinking water was significantly reduced.The hair was reversed and the back was slightly bowed.and the mice in the 10~6 dose group had abdominal distension symptoms;Among the 10~5,10~6,and 10~7 dose groups,there were 2 mice with reddew of the conjunctiva;compared to the?10~2 concentration group and the blank group had no abnormalities.After 15 days,the acute phase of each infected group was resistant.The mental state,activity,and drinking water of the surviving mice approached normal.However,the ToxoDB#9strains10~5,10~6,10~7 high dose group had 2,4,and 3 mice with limbs and neck masses;the?10~4 dose group and the blank group mice grew normally,and no abnormalities were observed.1.2 The infection rates of T.gondii in mice of 10~0,10~1 and?10~2 dose groups were 10%(2/20),40%(8/20)and 100%(20/20),respectively.The 10~0,10~1 group and blank group mice did not die;but?10~2 dose group could cause death in the experimental mice.1.3 The results of serum IgG antibody detection by ELISA showed that on the 3rd day after infection,the serum IgG antibodies of mice in infected group,RH strain group and blank group were all negative.On the 5th day after infection,the serum of IgG antibody was positive in ToxoDB#9 10~6group,while the RH strain,blank group and other infection groupswere all negative.On the 10th day of infection,the results of the 10~5,10~6,10~7 ToxoDB#9 dose group and the RH strain group were positive,and the other groups were negative.On the 20th,30th,and 45th days after infection,except for the 10~0 dose group and the blank group,the serum of the other infected groups were positive for IgG antibody.1.4 The results of PCR detection T.gondii traits B1 gene showed that the mice in the RH group and the?10~4 dose group were positive for PCR on the 5th day of infection,while the blank group and?10~3 dose group were negative;On the 10th day after infection,the test results of the?10~3dose group and the RH strain group were positive,and the other groups were negative;On the 20th day after infection,the blank group and the 10~o?10~1 dose group were negative,and the RH group and the other dose groups were positive;On the 30th and45th day of infection,the test results were the same:except for the 10~0 dose group and the blank group,the other infection groups were all positive.1.5 The results of serum cytokine test showed that the serum cytokines IFN-?level of each infected group was higher than that of the blank group,and the dose increase value increased.It reached the highest at 7 days after infection,and then decreased to the initial level of infection.The low-dose group gradually increased after 15 days of infection.The low-dose group,the middle-dose group,the blank control group and the RH group had smaller fluctuation range,and the high-dose group had larger fluctuation range.The high-dose group had higher serum IL-2 level than the other groups,and reached the highest level at 7days after infection.After that,it drops slightly and then rises again.The low-dose group,the middle-dose group,the blank control group and the RH group had lower fluctuations in IL-2levels.The medium-dose,high-dose,RH group mice had higher serum IL-12 levels than the blank group and low dose.In the group,the middle dose group RH strain control group reached the highest 7 days after infection,and the high dose group peaked at 15 days after infection.1.6 In the infected group,the naked eye observation combined with pathological section showed that the organs of the 10~5,10~6,and 10~7 dose groups had different degrees of damage,and the 10~6 dose group was the most typical:splenomegaly,congestion;renal enlargement;Hepatomegaly,congestion,liver degeneration,necrosis;lung swelling and consolidation,pneumonia;lung and kidney pathological section microscopic examination,alveolar widening,renal tubular epithelial shedding necrosis.2.In the second part of the study,the total positive rate of 311 fetal umbilical cord blood serum T.gondii IgG antibody in the obstetrics and gynaecology department of Mojiang County People's Hospital was 17.4%(54/311),including Hani,Han,Yi and Lahu.The positive rates of T.gondii IgG antibodies in other ethnic groups were 17%(33/194),18.1%(13/72),15%(3/20),25%(2/8),and 17.6%(3/17).There was no significant difference in the seroprevalence of T.gondii IgG antibody between different ethnic groups,neonatal gender,maternal age and pregnancy history(P>0.05).Conclusion1.Compared with RH strain,T.gondii from pigs in Dal infected Kunming mice with weak virulence in the RH strain.2.The total positive rate of T.gondii IgG antibody in Mojiang County of Yunnan Province was 17.4%. |
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