Cloning and Sequence Analysis of Toxoplasma gondii GRA25 Gene

In this study,sequence variation of GRA25 genes among 22 Toxoplasma gondii (T.gondii) strains from different hosts and geographical locations were examined.The complete GRA25 genes from 22 T.gondii isolates were amplified,sequenced,and nucleotide variations were determined.Phylogenetic analysis among the different T.gondii isolates were conducted using maximum parsimony (MP) and maximum likelihood (ML) methods.The biological characteristics of the protein GRA25 of the T.gondii RH strain were predicted using bioinformatics software.The sequences of all the examined T.gondii strains were 939 or 948 bp in length.Sequence comparison of all 22 GRA25 sequences identified 82 variable nucleotide positions (0 to 4.4%).The results of phylogenetic analysis showed that strains belonging to the classical typeⅠand Ⅱ could not group into their own branches based on the GRA25 sequences.Bioinformatics analysis revealed that the protein GRA25 contained 7 hydrophobicity regions,10 alpha regions,3 beta sheets,8 random coils and 8 linear B-cell epitopes.These results suggested that the GRA25 gene was not an ideal genetic marker for population genetic study of T.gondii strains,but it might represent a good vaccine candidate against toxoplasmosis.     

四种中药体外抗弓形虫效果的比较

目的:通过黄芩、甘草、马齿苋、桦褐孔菌四种中药体外抗弓形虫效果的比较,探讨其体外抗弓形虫的效果。方法用弓形虫RH株的速殖子感染培养的非洲绿猴肾细胞(VERO),加受试药物,以蒿甲醚为阳性药物对照,在加药后的12h、24h、48h观察细胞和弓形虫的生长情况及形态。加药48h后,分离出虫体,台酚蓝染色,计数,判断药物作用效果。结果:黄芩、甘草体外可明显抑制弓形虫的生长和繁殖,作用效果与药物浓度呈正相关;而马齿苋及桦褐孔菌体外抑制弓形虫效果较弱。     

Serological survey for antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in pet rabbits in eastern coastal areas of China

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly detected in rabbits and can infect humans and cause encephalitozoonosis. And Toxoplasma gondii is a prevalent parasite distributed worldwide and can infect almost all warm-blooded animals, including humans. The aim of the current study was to investigate the seroprevalence of E. cuniculi and Toxoplasma gondii, and risk factors of infection in pet rabbits reared in eastern coastal areas of China (Tianjin, Shandong, Jiangsu, Zhejiang, Shanghai and Fujian). Total 222 blood samples of pet rabbits were collected from local veterinary hospitals. The seropositivity rates of E. cuniculi were 16.22% (36/222) according to an Enzyme-linked immunosorbent assay (ELISA). Female pet rabbits was significantly higher than that in males (P=0.002), Zhejiang were markedly higher than those in Jiangsu and Shanghai (P=0.017, P=0.022), and cross-breed rabbits were dramatically higher than those in Chinchilla, New Zealand white, Rex (P=0.02, P=0.006, P=0.008). The seroprevalence of T. gondii was 13.06% (29/222) by the method of ELISA. The seroprevalence in Zhejiang was significantly higher than that in Shanghai (P=0.017). No difference in seroprevalence was detected with respect to the gender, age, species, health status, or season. These findings show that E. cuniculi and T. gondii are present and spread in pet rabbits. Therefore, pet rabbits should be considered as an important reservoir of encephalitozoonosis for humans and maybe important implication for public health in eastern coastal areas of China.     

Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at −30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.     

抗弓形虫和新孢子虫药物体外筛选方法的研究进展

弓形虫和新孢子虫是2种在形态结构及生物学特征方面非常相似的细胞内寄生性原虫,二者引起的疾病对人和动物健康造成了严重危害。开发和筛选高效、低毒的抗弓形虫和新孢子虫药物一直是抗原虫药物研究领域的重要方向之一。药物筛选包括体外筛选和体内筛选,体外筛选的结果是进行体内筛选的依据和基础。对抗弓形虫和新孢子虫药物的体外筛选方法研究进展进行综述,以期为开发更加高效的抗弓形虫和新孢子虫药物体外筛选技术提供参考。