| Toxoplasmosis is a widespread and harmful zoonosis. This disease is capable of infecting human and almost any warm-blooded animal. The infection to adults mostly is inapprent , but pregnant women infected with toxoplasmosis could lead to abortion, weak child, or stillbirth . Besides, the disease is also seriously harm to pregnant livestock production and cause serious economic losses to animal husbandry. So it is essential to establish a set of scientific diagnostic methods and effective treatment measures for toxoplasmosis.The surface protein (SAG1 and SAG2) and rop-like protein of Toxoplasma gondii are considered to be the two candidates with great advantages in the diagnosis and vaccine research of toxoplasmosis. ROP18 could expresse in the stages of Toxoplasma gondii tachyzoites and bradyzoites, which is a serine-threonine kinase with kinase activity and had highly immunogenicity. The study about it are reported rarely at home and abroad. In this study, we first designed two primers according to the known gene sequence and extracted the total DNA of Toxoplasma gondii RH strain as template, then amplified the major gene of ROP18 by PCR. The truncate ROP 18 gene was legated to prokaryotic expression vector pET32a(+). The recombinant plasmids pET32a(+)-rop18 was constructed and transformed into BL21(DE3) for expression. Fusion protein was expressed as inclusion bodies and the expressed product after refolding had good immunogenicity according to western blot analysis. Using purified rROP18 and rSAG2 recombinant protein, the initial ELISA method was established for detecting Toxoplasma gondii in dogs, and could detect antibodies in the serum of different stages of Toxoplasma gondii (acute, chronic).In the detection of the 72 dogs'serum, the results of positive Rate was 12.5%, and coincidence rate with IHA positive result was 85.7%, negative coincidence rate was 95.4%.At present, the treatment of toxoplasmosis was also dependent on drugs; the known effective medication of anti-Toxoplasma gondii had large toxic side effects, plus the lack of animal dedicate anti-Toxoplasma drugs, so it was urgent to look for an efficient, low toxicity dedicated anti-Toxoplasma drugs in animal.Traditional Chinese drugs not only had the advantage of low toxicity and low residue, but also could improve animals immunity and have curative effect of reducing thermal and so on. So this study attempted to screen the mixture of medicine and Traditional Chinese drugs, and use the fast and effective role of medicine combining with the advantage of traditional Chinese drugs( cooling and regulate the , low toxic side effects ) to treat toxoplasmosis.Using the tachyzoite of T. gondii RH strain infecting the Ana-1 cells in vitro, determined the safe concentrations of drugs on cell concentration by MTT assay, and then added the different kinds of the Chinese herbal medicine. About 4, 8, 12, 24, 48 hours after adding medicine, the growth condition and the configuration of the cells and T. gondii were observed respectively. Drug effects in inhibition of parasite proliferation in vitro were estimated after adding the drugs 48 hours. The results indicated that Sulfonamides chlorpromazine sodium had better inhibitory effect than sulfadiazine sodium, and glycyrrhiza, scutellaria and stemonae could significantly inhibit the proliferation of T. gondii, in vitro. The combination of the traditional Chinese drugs and medicines for anti-Toxoplasma was validated by treating KM mice which were infected with Toxoplasma gondii.The results showed that scutellaria and glycyrrhiza uralensis could significantly prolong the time of death, and sulfonamides chlorpromazine sodium could make 50% of mice infected with toxoplasma survival, and the drug scutellaria and glycyrrhiza uralensis were formed combination drugs,The results of this experiment suggested that the combination could reduce the dose of medicine based on keeping good therapeutic effect.The effective combination of sulfonamides chlorpromazine sodium, scutellaria and glycyrrhiza uralensis was primarily administrated to 40-days old pigs which were infected with toxoplasma artificially. The symptom of fever could be controlled quickly. The time of blood with toxoplasm and the peak values of circulating antigen (CAg) in serum were lower than positive control group. The speed of worm-emia and CAg of serum changed to be negtive faster than positive control group. The results indicated that combination of sulfonamides chlorpromazine sodium, scutellaria and glycyrrhiza uralensis could effectively treat toxoplasmosis with little side effect under therapeutic dosage. |
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