Toxoplasmosis is one of the most common zoonotic diseases in the world. Felines excrete environmentally resistant Toxoplasma gondii oocysts. However, there is no direct evidence to prove tigers are the intermediate host of T. gondii. Here, we show that, IgG antibodies to T. gondii in 80% (8/10) of captive tigers. Two viable T. gondii strains (ToxoDB genotype #9) were isolated by bioassay in mice using striated muscles of two tigers (Tiger#3 and Tiger#8). Additionally, mice were confirmed as T. gondii-positive by bioassay of feces #89–110, but no viable T. gondii strain was isolated successfully. The fecal samples from tigers may contain T. gondii oocysts. This is the first report of T. gondii isolation from tigers. These results provide direct evidence that an extra-intestinal cycle of T. gondii may develop in tigers.

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

Sarcocystis is an important zoonotic protozoan parasites. This study collected 354 tissue samples (22 skeletal muscle samples, 74 diaphragm samples, 171 heart samples, and 87 esophagus samples) from 354 sheep across seven slaughterhouses in seven cities of Xinjiang. Screening for Sarcocystis spp. was performed using PCR targeting the 18S rRNA gene, with confirmation PCR targeting the COX 1 gene. The infection rate was determined to be 30.2% (107/354). All seven cities tested positive for Sarcocystis, with the highest infection rate in Tacheng (95.5%, 21/22) and the lowest infection rate in Kuqa (9.2%, 9/98). The infection rates by tissue were as follows: 95.5% (21/22) in skeletal muscles, 40.5% (30/74) in diaphragm, 24.0% (41/171) in heart, and 17.2% (15/87) in esophageal, respectively. Two Sarcocystis species were identified from the 107 positive samples: S. tenella (n = 106) and S. arieticanis (n = 1). Five genotypes of S. tenella and one genotype of S. arieticanis were identified, all of which clustered closely with previously discovered isolates. These results enhance our understanding of the epidemiological status of Sarcocystis in sheep in Xinjiang.

The transport mechanisms for substrates and nutrients within the heme pathway of the Toxoplasma gondii apicoplast remain poorly understood. However, studies on heme metabolic enzymes have employed disparate genetic manipulation approaches, limiting direct phenotypic comparisons among different enzymes. This research involved screening potential apicoplast proteins in Toxoplasma gondii by cross-referencing and analyzing protein–protein interaction networks. Within the heme enzyme pathway of the apicoplast, eight enzymes were found to be predominantly conserved in the Sarcocystide family. Utilizing the CRISPR-Cas9 system alongside a U1 snRNP-mediated gene-silencing approach, we developed inducible knockdown strains—iKD-PBGD, iKD-UROS, and iKD-UROD—targeting three key metabolic enzymes crucial for the parasite lytic cycle, as demonstrated through replication experiments. To investigate the transport mechanisms for heme-related nutrients or substrates, we knocked down these three enzymes, using TgGRA12 as an initial marker. Continuous fluorescence signals highlighted the parasitophorous vacuole (PV) membrane surrounding tachyzoites during both early and late replication stages, particularly at 48 h post-rapamycin treatment, indicating a transformation of the cyst-like PV resembling that in Toxoplasma gondii. Phenotypically, knockdown of these heme enzymes led to the formation of slowly replicating, cyst-like parasitophorous vacuoles. However, this morphological change did not significantly affect the acute virulence of the parasites in vivo, as determined by mouse survival assays. This study explored the functional roles of the three intermediate metabolic enzymes, offering a novel viewpoint on the gradual demise of Toxoplasma gondii as a potential target for drug development.