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Simple Summary: Toxoplasmosis is one of the most common parasitic zoonoses in the world. It not only threatens human health and safety but also causes considerable losses to the global animal husbandry industry. Swine, as an intermediate host of Toxoplasma gondii, has important economic and public health significance, and the global prevalence of T. gondii in swine varies between 10% and 60%. Therefore, it is important to establish simple, effective, sensitive and specific diagnostic methods for the detection and prevention of T. gondii infection in swine. In the present study, we developed a colloidal gold immunochromatographic strip based on rTgROP14, recombinant protein A and monoclonal antibody TgROP14-5D5 for serological detection of T. gondii in swine populations. This new ICT achieved good specificity and sensitivity and has potential for routine serological detection of T. gondii in swine. Toxoplasma gondii, a worldwide distributed apicomplexan protozoan, can infect almost all warm-blooded animals and may cause toxoplasmosis. In order to provide a point-of-care detection method for T. gondii infection, an immunochromatographic test (ICT) was established. The proposed test uses recombinant T. gondii rhoptry protein 14 (ROP14) conjugated with 20 nm gold particles, recombinant protein A as the detection line and monoclonal antibody TgROP14-5D5 as the control line. The specificity, sensitivity, positive predictive value, negative predictive value and stability of this new ICT were evaluated. rTgROP14 was specifically recognized by positive serum of T. gondii but not negative serum. mAb TgROP14-5D5 showed higher specific recognition of T. gondii antigens and was therefore selected for subsequent colloidal gold strip construction. The new ICT based on TgROP14 exhibited good diagnostic performance with high specificity (86.9%) and sensitivity (90.9%) using IHA as a "reference standard". Among 436 field porcine sera, ICT and IHA detected 134 (30.7%) and 99 (22.7%) positive samples, respectively. The relative agreement was 87.8%. These data indicate that this new ICT based on TgROP14 is a suitable candidate for routine testing of T. gondii in the field. [ABSTRACT FROM AUTHOR]

The apicoplast, which is the result of secondary endosymbiosis, is a distinctive subcellular organelle and a crucial therapeutic target for apicomplexan parasites. The majority of apicoplast-resident proteins are encoded by the nuclear genome and target the apicoplast via bipartite targeting signals consisting of a signal peptide and a transit peptide. The properties and functions of these peptides are poorly understood, which hinders the identification of apicoplast proteins and the study for plastid evolution. Here, the targeting signals of the recently discovered apicoplast tRNA thiouridylase Tg MnmA of Toxoplasma gondii were analyzed. Our data using a reporter (the enhanced green fluorescent protein) fused with individual fragments containing various numbers of its N-terminal amino acids unequivocally revealed that the first 28 amino acids of Tg MnmA functioned as a signal peptide for cellular secretion. The N-terminal 150 amino acids were sufficient to direct the fusion protein to the apicoplast, whereas its deletion caused the fusion protein to be localized to the mitochondrion. Our data further demonstrated that the apicoplast, rhoptry, and mitochondrion shared similar targeting signals, indicating that the apicoplast localization peptide was trans-organellar in function. In addition, the apicoplast localization peptide was important for the healthy proliferation of tachyzoites. In conclusion, the targeting signals of the nucleus-encoded apicoplast-targeted protein Tg MnmA have been mapped out and the importance of this localization peptide has been elucidated in the current study. • The first N-terminal 150 AA of Tg MnmA target the EGFP to the apicoplast. • Without the localization peptide, Tg MnmAΔ1−150-EGFP localizes to the mitochondrion. • Mis-localization of Tg MnmA greatly affects the proliferation of T. gondii in vitro. [ABSTRACT FROM AUTHOR]