Isolation And Biologic Characterization Of Two Toxoplasma Gondii Strains From Pigs

Toxoplasma gondii is an obligate intracellular protozoan that belongs to the phylum Apicomplexa, class Sporozoasida, order Eucoccidia, family Toxoplasmatidae, genus Toxoplasma. T.gondii infections are prevalent in humans and animals worldwide. Felids are the definitive host of T.gondii, humans and more than200kinds of animals are the intermediate host. Toxoplasmosis is very dangerous to humans and animals. Infection acquired during pregnancy may cause severe damage to the fetus, causing premature delivery, abortion and fetal abnormality. Toxoplasmosis is the one reason that causing monstra and also the major reason for death of immunocompromised patients, such as AIDS patients. Animal infection is common, and mostly do not take on clinical symptoms. However, the pigs are widely infected with high mortality, which causes significant economic losses to pig industry.The transmissions of T.gondii are congenital infection and postnatally acquired infection. Humans become infected post-natally by ingesting tissue cysts from undercooked meat. Pork is the main meat consumed by chinese people. Also, pig industry is a major sector of the livestock industry in China. The pork contaminated with T.gondii may be the important source of infection for human toxoplasmosis. So, it’s very important to carry on a research about T. gondii isolates from pigs, which contributes to recognize the prevalence of T.gondii in pigs. It has significant implications for the control and dignosis of pig infection with T. gondii. Also, the research may help us master the transmission of T.gondii and formulate the strategy in public hygiene.1. Establishment of a specific PCR to detect T.gondiiICR mice was inoculated with T.gondii RH strain via abdominal cavity. After3to5days of inoculation the ascites of mice were collected and T. gondii tachyzoites were purified through CF-11cellulose column. The DNA of T. gondii tachyzoites were extracted with general method. A specific PCR to detect B1and529repeated genes of T.gondii were established. The results showed that the minimum numer of tachyzoite to be detected was about0.9by529-PCR,9by B1-PCR and90by duplex-PCR.2. Isolation and detection of T.gondii from pigsT.gondii in tissues of pigs was detected by the PCR method established in the first part. The tissue samples were collected from pigs taking on the clinical symptoms, such as high temperature and breathing difficulties; the liver, lungs, lymph nodes and intestinal tract had hemorrhagic spots and necrosis with necropsy.5positive samples were detected in25samples, the positive rate was20%. Then the positive samples were used for isolating T.gondii by ICR mice. The dead mice were necropsy to collect ascites. The ascites were stained to find tachyzoites and pseudocysts. Two strains were successfully isolated from the5positive samples, and the success rate was40%. The two strains were named as YZ-1and YZ-2, they were isolated from domesticated wild boar and domesticated pig respectively.3. The mouse-virulence analysis of two T.gondii isolates from pigsThe two T.gondii strains isolates were keeped on inoculating in ICR mice with the same numbers every time until the tachyzoites propagated steadily in abdominal cavity. The RH was selected as reference strain, each strain had5groups. One group was negative control group. Then the tachyzoite of each strain was diluted into five different does and inoculated ICR mice via abdominal cavity. As a results, the occurring regularity of death of the two isolates was unanimous to RH strain, so we recognized the two isolates were high virulent to mice. However, the GRA6gene sequences of the two isolates were completely identical to the less virulent strain-BEVERLEY, which all of them had15bp and3bp deletion.4. Genotype of the two T.gondii isolates from pigsThe two T.gondii isolates were genetically characterised using nine PCR restriction fragment length polymorphism markers including SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1and Apico. The result performed that the genotype of the two T.gondii isolates was identical to the genotype Chinese1. In addition, the phylogenesis trees of GRA6, SAG2and PK1genes were described to analyse the genotype of the two isolates, and the result showed that YZ-1and YZ-2strains belonged to genotype II branch.5. ITS sequences analysis of the two T.gondii isolates from pigsThe ITS and5.8S sequences of the two T.gondii isolates were analysed, and the results suggested that the sequences of ITS among T.gondii strans were highly similar and conservative. These datas provided a theoretical basis for finding a kind of fast and effective method to detecte T.gondii. Also, the N-J phylogenesis trees of ITS and ITS1sequences were constructed with other protozoan, and the result showed that ITS sequence was appropriate to distinguish T.gondii from other protozoan, but not useful to study the intraspecific genetic variation of T.gondii.