Studies On The Transmission Of Toxoplasma Gondii By Semen In Rabbits

Toxoplasmosis, caused by Toxoplasma gondii, is widespread in man and warm-blooded animals. Although usually asymptomatic in immunocompetent individuals, toxoplasmosis may cause severe disorders in immunocompromised patients and in pregnant women because of the high risk of transplacental transmission and the occurrence of multiple congenital lesions in the fetus . In animal husbandry, it can cause severe economic expenses by the animal's disease and abortion. It is endemic worldwide and, depending on the eographic location.The complex life cycle and many routes of infection were found with Toxoplasma gondii. Peoples can be infected with it by eating row or uncooked meals or by drinking water. When the initial infection take place with the pregnant women, foetus can be infected via placenta. The tachyzoites of Toxoplasma gondii can get into the vivo by the wound . It can be transmitted by blood transfusion. The case had been reported that infection emerging after the cat' biting.In recent years,the Toxoplasmosis distribution features had been found that infection rates of peoples in west were higher than in underdeveloped regions. The tendency also had been found that it increased up year by year in China,the domestic average infection rate of Toxoplasma gondii in 1985 —1996 was 4.9%,but it was 7.88% in 2004.Eating more meats in Chinese may causes the increasing of infection rates ,but it isn't known that more chances of extramarital sexual intercourses if affect the infection rates.The Toxoplasma gondii can parasites in all host's karyocytes besides red blood cell.The frequent damaged organs are consisted of brain,eye,heart,liver,lung , kidney and so on. The tachyzoites of Toxoplasma gondii had been found in testicle and semen. The DNA fragment of Toxoplasma gondii had been detected in vagina by PCR. The topic has not been deeply probed that the Toxoplasma gondii if is transmitted by sexual intercourse and the transmission if is affected by the healthy states of vagina.For tha past years,because the tachyzoites of Toxoplasma gondii were very tiny and the infection of it often appeared in suppressive state, so the detection of it was relyed on serodiagnosis which was belong to qualitative test.The etiologic diagnosis of Toxoplasma gondii was carried out only by mice inoculation.The kinetics of tacyzoites in host have not been investigated up to now.That the real-time PCR established in 1996 and the utilized in quantitative detection of Toxoplasma gondii by it in 2000 made it possible to analyse the kinetics of tachyzoites in host. In our study, the animal models were established with the male rabbits infected by RH strain Toxoplasma and female rabbits made in different healthy status of vagina. The kinetics of tachyzoites in blood and semen from infected male rabbits were analysed with conventional PCR and real-time PCR. The Toxoplasma gondii infection of female rabbits after insemination with semen from infected male rabbitd were detected by ELISA and conventional PCR. Part one:Construction of the animal model in the male and femalerabbitsTo construct the Toxoplasma gondii infection model in the male rabbits and the female rabbits models with different vagina states, 16 healthy male rabbits were infected with T. gondii by intraperitoneal injection with 1×10⁵ RH tachyzoites for male model establishment. A self-make false vagina was used to collect a male rabbits' semen weekly after infection. The samples of blood were collected from the same male rabbit's helix vein at the same time. 27 health female New Zealand rabbits , Body weight of every rabbit was about 3 kilograms , were divided into 4 groups randomly for female rabbits model establishment, and there were 7 in every group except 6 in the 4^th group. Group 1 was normal vagina group; Group 2 was injury vagina group, a sterilized cotton swab was inserted into the vagina and brushed until bleeding; Group 3 was trichomoniasis vaginitis group, 1ml trichomoniasis vaginitis trophozoites liquid was dropped into the vagina every day. The criterion of trichomoniasis vaginitis was that trophozites could be discovered in the vagina excreta film by optical microscopy and Group 4 was colpomycosis group, 1ml of bacterium liquid was injected into vagina for 3 times continuously . The criterion of colpomycosis was the appearance of white color plaque by naked eye.Results: male rabbits: 16 healthy male rabbits were infected with T. gondii by intraperitoneal injection with 1×10⁵ RH strain tachyzoites. 8 out of 16 rabbits died on 8～14d after infection. In the alive male infection rabbits ,the blood samples could be collected weekly. But due to Toxoplasma gondii infection , the semen samples couldn't be harvested in the period of 7-14 days after infection. Femal rabbits model:The four different vagina health states or female rabbits were established successfully.Conclusion: The half of 16 male rabbits died of T. gondii RH strain infection. The sample collection from semen was more difficult than that from blood. The four vagina health states in female rabbits were similar to human being's.Part two:The establishment of immunological assay for detection of antibody against Toxoplasma gondii in sera from rabbits.To establish Enzyme-linked immunosorbent assay for detection of toxoplasma antibodies IgG from the sera of rabbits,the purified trophozoizes were treated with three cycles of freeze-thawing and ultrasonication and subjected to ultracentrifugation. The supernatant was as the antigen for coating the well of ELISA microhaemagglutination plates. 100ul of mixed positive sera of rabbits were diluted and dropped into the well of the plates. The optimal dilution of antigen and sera in ELISA were determined by checker board titration. In the ELISA assay, the goat anti-rabbit antibody labeled with HRP was as the 2nd antibody and the OPD as the substrate.Results: The optimal protein content of antigen coated was 0.15ug/ml and optimal sera dilution was 1:80 in ELISA.Conclusion: The establishment of ELISA for IgG antibody against Toxoplasma gondii in sera from infected rabbits was successful.Part threeChoosing the optimal conventional PCR assay system for detection of Toxoplasma DNA in body fluids from infectedrabbitsTo evaluate the PCR kits developed and produced by domestic corporations, the optimal Kits for detection the Toxoplasma gondii DNA in blood and semen from infected rabbits were picked up. 4 types of kits from 4 corporations(Co) were enrolled into this research for toxoplasma detection in blood or semen from rabbits and all detections were preformed as their directions indicated. Results: No positive result could be detected in semen or blood samples from rabbits either before or after infection by TOX-PCR kits from Weizhou Yilikang Co. and Beijing MDC Co.; All samples were negative before infection and turned to be positive after infection when they were detected by TOX-PCR kits fromtoShanghai Fusheng Co, but there were too much non-specific bands; All samples were negative before infection, but 13 out of 17 blood samples and 1 out of 43 semen samples turned to be positive when kits from Luoyang Sino-Ame Co were used.Conclusions: The practical application of TOX-PCR kits marketed in China was very limited. TOX-PCR kits from Shanghai Fusheng Co were suitable for detection of Toxoplasma DNA in semen from infected rabbits. That from Luoyang Sino-Ame Co was suitable in blood.Part four:The establishment of fluorescent quantitation PCR for quantitative detection of Toxoplsama gondii DNA inrabbitsTo establish the fluorescent quantitation PCR using the TaqMan probe for quantitative detection of Toxoplasma gondii DNA in blood and semen in rabbits,the primer in this FQ-PCR was targeted at the T. gondii Hsp20. Hsp20 gene amplicon comprises a 240-bp fragment. The primers used for amplification were the 5'-GCGACATGGACGAGATGCT-3' (sense) and the 5'-CAGTTGGCCTTCGCCGACC-3'. (antisense).The extraction of toxoplasma gondii DNA was practiced by heating the semen samples with base. The extraction of DNA from blood was with NaI. The FQ-PCR was preformed with the PE7000 Detection System (PE Applied Biosystem, U.S.A.). Results: All of 8 blood samples before infection were FQ-PCR negative .7 blood samples after 1～3 weeks infection were FQ-PCR positive. 6 semen samples before infection were negative.3out of 5 semen samples could be quantitated with some densities of trophozoites by FQ-PCR.Conclusion: The sensibility and specificity of FQ-PCR assay established could be satisfied with quantitive detection of the samples from infected rabbits.Part five:Kineses of Toxoplasma gondii in Semen and Blood frominfected RabbitTo observe the kinesis of Toxoplasma gondii in Semen and Blood from Infected Rabbit and to compare the dynamoic detection of Toxoplasma gondii DNA in blood bytwo PCR, each of 16 male rabbits was infected with 10⁵ RH strain toxoplasma tachyzoites by abdominal inoculation. The semen and blood collected before and after infection were immediately stored at -40°C until using. Conventional PCR was used to detect the Toxoplasma gondii DNA .Fluorescent quantization PCR (FQ-PCR) was used to quantitate the DNA copies of Toxoplasma gondii and the densities of Toxoplasma gondii were calculated from the standard curves. Then the dynamic changes curve of densities of Toxoplasma gondii in semen or blood were set up. Results: The quantitative detection of Toxoplasma gondii DNA by FQ-PCR: The all blood samples before infection were FQ-PCR negative.The positive rate of all blood samples after infection was 64.42%.The peak of densities of Toxoplasma gondii in blood was appeared in about 1 week after infection. The maximum density of Toxoplasma gondii was 2.48 × 10⁵ trophozoites /ml in blood,then it was kept low level.The minimum density of Toxoplasma gondii in blood was 1.45 ×103 trophozoites / ml on the 121^th day after infection. The all semen samples before infection were FQ-PCR negative ,the positive rate of all semen samples from the infected rabbits was 26.67%. The primary positive in semen appeared on 7^th day after infection.The maximum density of Toxoplasma gondii in semen was 1.48 × 105 trophozoites / ml.The peak in semen was appeared in 7^th week after infection,after the peak,the densities were kept in plateau level.The densities of 4.68 ×10³ trophozoites / ml in semen still were quantitated in the 15 week after infection.The qualitative detection of Toxoplasma gondii DNA by Conventional PCR:The total positive rates of Toxoplasma gondii DNA in blood from infected rabbits were 45%.The positive rate only was 22% in first week after infection. The peak of positive rate was kept in 2～4 week after infection, in which the positive rate was about 60%,then it in semen was kept about 22%.The primary positive reaction in semen by this PCR was appeared on the 8^th day after infection,still there was positive reaction in semen on the 72^th day after infection.Conclusion: The positive rates kinesis of toxoplasma gondii DNA detections in blood from infected rabbits by conventional PCR was similar to that by FQ-PCR. The optimal time of toxoplasma gondii DNA detection in blood by PCR was kept in 1～4 week after infection, then the positive rates was fall-off. The kinesis of toxoplasma gondii in blood from infected rabbits was different with that in semen.Part Six:Studies on the Transmission of Toxoplasma gondiiby Semen in RabbitsTo confirm the transmission of Toxoplasma gondii (T. gondii) by semen and to investigate the impact of vaginal status on the transmission of T. gondii in female rabbits, 16 male rabbits were infected with T. gondii by intraperitoneal injection each with 1 × 105 RH tachyzoites. 8 out of 16 rabbits died on 8-14 d after infection. Artificial vagina was used to collect semen from male rabbits weekly before and after infection for 8 weeks. If more than 2 portions of semen from 8 survived male rabbits were collected after infection, the collected semen was mixed weekly for later use. 27 female rabbits were divided into 4 groups: group 1 with normal vagina (7 rabbits), group 2 with wounded vagina (7), group 3 with Trichomona vaginitis (7) and group 4 with colpomycosis infection (6). Tachyzoites were found in mixed semen digested by trypsinase, and were used for endovaginal artificial insemination to female rabbits by uterine cavity tube once a week for 8 consecutive weeks. 2～3 d after every insemination, 2 ml blood was collected from helix vein of each rabbit, and the blood samples were stored at -40°C for use. Anti-I gondiiantibody was examined by ELISA and the B1 gene of T. gondii was detected by PCR.Results :Anti-T. gondii antibody was detected in some rabbits (2, 3, 1, and 1 rabbits from each of the groups respectively) on the 16th day after the first insemination. The positive rate of ELISA was 25.9%. The amplification of B1 gene (200bp) by PCR appeared positive from the blood samples on the 3rd day after the first insemination and the last positive one was proved on the 51th day after the first insemination. Number of positive samples was 2, 1, 3 and 1 in the 4 groups respectively, with an over all PCR positive rate of 18.5%. Only 3 of the 27 rabbits were positive by both ELISA and PCR.Conclusions :T. gondii can be transmitted by semen and the health status of vagina shows no impact on it.Conclusions were obtained from above experiments: When each of 16 male rabbits was infected with the dose 1×10⁵ tachyzoites of RH train Toxoplasma gondii,8 out of them died of infection in 7～14days after infection.The 4 health status model of vagina was established with 27 female rabbits. The optimal detection time was within 1～4 weeks after infection, subsequently the detection rates decreased, when the DNA of Toxoplasma gondii in blood was detected with conventional PCR and real-time PCR. When the DNA of Toxoplasma gondii in blood and semen from the infected rabbits was continuously detected by fluorescent quantitation PCR, the kinetics in blood and in semen were different, the peak of Toxoplasma gondii density in blood was within 1 weeks after infection, but it in semen was within 7weeks after infection.The toxoplasma transmission rate by semen from infected male rabbits was 11.1%, the health status of vagina in 4 groups female rabbits had no influence on the transmission rates ,when the infection of toxoplasma gondii with 27 female rabbits after insemination was detected with ELISA or PCR .