The Establishment Of PCR Assay For Diagnosis And Safety And Immunity Efficacy Of NTA Attenuated Strain Of Toxoplasma Gondii

Toxoplasma gondii is an obligate, intracellular protozoal infecting humans beings, many warm-blooded animals and birds, classified in Eucoccidiida. Individuals with normal immunity have frequently persistent infection. Individuals with impaired immunity and those with acquired infection are particularly susceptible, e.g., those infected with the human immunodeficiency virus and persons accepting transplanted organs. Toxoplasma gondii infection is highly prevalent in pigs in most countries. Most adult pigs acquire frequently subclinical infection. Clinical toxoplasmosis occurs mostly in young pigs which are most susceptible. Transplacental infection appears to be less common than post-natal infection. At presently, the vaccine of toxoplasmosis in animals is not available in China. The diagnostic methods frequently used for detection T.gondii infection of animals are clinic diagnosis, pathogenic diagnosis and IHA test which have limitations in specificity, sensitivity and early diagnosis. ITS1-PCR assay for detecting T.gondii was developed and the safety and immunity efficacy of NTA attenuated strain was evaluated in this study.We aimed to establish a specific and sensitive serial of diagnosis methods for detecting T.gondii in swine. The PCR assay targeting multicopy 18S-5.8S rRNA internal transcribed spacer l(ITS-l) region had first been developed for the diagnosis of toxoplasma infection in this test. Tachyzoites DNA was diluted and PCR amplified to detect the sensitivity of PCR assay. In order to detect its specificity Eimeria Tenella in Chicken was used as the control and amplification using the same primer pairs was performed with DNA from Geographic Eimeria Tenella in Chicken. Experimentally infected mice and rabbits and the clinical samples including lungs, bronchopulmonary lymph nodes, spleen, kidney and livers from 10 pigs with doubtful toxoplasmosis were detected by tissue smear method and PCR amplification to comply the sensitivity of these two methods DNA. Results: This assay was able to detect as little as 1pg of T.gondii genomic DNA, which is equivalent to 10 T.gondii tachyzoites. The Results obtained by the specific test showed only T.gondii had a specific band (300bp) targeting ITS1 region, while no amplification was seen with DNA...