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Fatal infections with the rare COUG strain of the zoonotic parasite Toxoplasma gondii were recently detected for the first time in four southern sea otters ( Enhydra lutris nereis ) exhibiting severe protozoal steatitis. The objectives of this study were to describe new COUG strain infections in sea otters, investigate the potential contributory role of a recently discovered parasite-infecting narnavirus ( Apocryptovirus odysseus ) in these infections, assess the potential contribution of vitamin E deficiency in the development of systemic steatitis, and explore the utility of serotyping for strain-specific diagnosis of T. gondii infections in sea otters. Since initial reporting, six additional sea otters died due to fatal COUG strain T. gondii infections. Five animals exhibited lesion patterns resembling the prior case definition including severe, widespread steatitis. The final case died due to severe T. gondii -associated meningoencephalitis with no grossly or microscopically apparent steatitis. In contrast with a recent report utilizing a cougar-derived parasite isolate, A. odysseus RNA was not detected in sea otter-derived COUG strain isolates, suggesting that this narnavirus is not associated with fatal COUG strain infections in sea otters. Serotyping using dense granule (GRA) peptides to distinguish between T. gondii strains infecting sea otters suggests that Type X, Type II, and COUG strains exhibit different peptide-reactivity profiles that may allow them to be distinguished serologically. COUG strain T. gondii infections are an emerging threat to southern sea otter population health, and this strain has the potential to infect other animal and human hosts that share their environment and food sources with sea otters. Additional studies are needed to clarify the environmental sources, epidemiology, pathophysiology, and premortem serodiagnosis of COUG strain T. gondii infections in southern sea otters and other susceptible hosts.

Toxoplasma gondii is a worldwide prevalent parasite, affecting a wide range of mammals and human beings. Little information is available about the distribution of genetic diversity of T. gondii infection in minks (Neovison vison). This study was conducted to estimate the prevalence and genetic characterization of T. gondii isolates from minks in China. A total of 418 minks brain tissue samples were collected from Jilin and Hebei provinces, northern China. Genomic DNA were extracted and assayed for T. gondii infection by semi-nested PCR of B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2 (5'+3' SAG2, alter.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. 36 (8.6%) of 418 DNA samples were overall positive for T. gondii. Among them, 5 samples were genotyped at all loci, and 1 sample was genotyped for 9 loci. In total, five samples belong to ToxoDB PCR-RFLP genotype#9, one belong to ToxoDB genotye#3. To our knowledge, this is the first report of genetic characterization of T. gondii in minks in China. Meanwhile, these results revealed a distribution of T. gondii infection in minks in China. These data provided base-line information for controlling T. gondii infection in minks.

Prior studies have demonstrated that B cells are important components of protection in vaccinated mice challenged intraperitoneally with a highly virulent type I strain of Toxoplasma gondii parasites. However, it is not known whether B cells are required for vaccinated mice to successfully resist a more physiologically relevant challenge infection with a mildly virulent type II strain of T. gondii. To investigate that question, we vaccinated B cell-deficient C57BL/6 (μMT) mice with an attenuated strain of T. gondii and challenged them with a potentially lethal oral dose of type II T. gondii cysts. Vaccinated μMT mice survived the challenge as well as vaccinated B6 controls, controlled parasites equally well in critical tissues, produced equivalent levels of mRNA for several type 1 cytokines, and exhibited comparably mild histopathology. Thus, a vaccine can protect against infection with a mildly virulent type II strain of T. gondii in the absence of a B cell-dependent immune response.

Key Points The parasite Toxoplasma gondii is extremely widespread in animals and is a common cause of food- and water-borne infection in people. Although most infections are benign, they can have severe consequences in immunocompromised patients and following congenital infection. T. gondii is regarded as a model intracellular parasite for which forward- and reverse-genetics tools are available. In combination with the mouse model of toxoplasmosis (including the many genetic knockout and transgenic mouse lines that are available), these tools for genetic manipulation of the parasite have enabled researchers to explore the molecular determinants of T. gondii pathogenesis and host defence. Forward-genetics crosses conducted in T. gondii , using strains of different genotypes and virulences in mice, revealed that acute virulence is largely mediated by a family of effector proteins that are secreted into the host cell cytoplasm during parasite invasion. These proteins are derived from a secretory organelle called the rhoptry and, hence, are called ROP effectors. ROPs include a family of serine/threonine kinases that affect host targets and have important roles in infection in the mouse. Among these, ROP18 phosphorylates immunity-related GTPases, thus promoting parasite survival in activated macrophages, whereas ROP16 phosphorylates signal transducer and activator of transcription 3 (STAT3) and STAT6 and, hence, alters host gene transcription. Curiously, the activity of ROP18 is mediated by another family member called ROP5, which is a pseudokinase. Although a limited subset of ROP kinases can largely explain the virulence of T. gondii in the mouse, their role in other hosts has not been established. The genome encodes more than 40 ROPs, and these different proteins might have distinct roles during infection in the wide range of hosts infected by T. gondii . Understanding these patterns might help in the prevention and treatment of human infections. The intracellular parasite Toxoplasma gondii can infect a range of hosts and occasionally causes serious disease in humans. In this Review, Hunter and Sibley summarize recent studies that implicate rhoptry kinases and a dense-granule protein as mediators of acute virulence in the mouse model. They also describe the complex interplay between these parasite effector proteins and the innate immune system. Toxoplasma gondii is a common parasite of animals and humans and can cause serious opportunistic infections. However, the majority of infections are asymptomatic, possibly because the organism has co-evolved with its many vertebrate hosts and has developed multiple strategies to persist asymptomatically for the lifetime of the host. Over the past two decades, infection studies in the mouse, combined with forward-genetics approaches aimed at unravelling the molecular basis of infection, have revealed that T. gondii virulence is mediated, in part, by secretion of effector proteins into the host cell during invasion. Here, we review recent advances that illustrate how these virulence factors disarm innate immunity and promote survival of the parasite.

Toxoplasmosis, caused by the obligate intracellular protozoan Toxoplasma gondii , is an important zoonosis with medical and veterinary importance worldwide. The disease is mainly contracted by ingesting undercooked or raw meat containing viable tissue cysts, or by ingesting food or water contaminated with oocysts. The diagnosis and genetic characterization of T. gondii infection is crucial for the surveillance, prevention and control of toxoplasmosis. Traditional approaches for the diagnosis of toxoplasmosis include etiological, immunological and imaging techniques. Diagnosis of toxoplasmosis has been improved by the emergence of molecular technologies to amplify parasite nucleic acids. Among these, polymerase chain reaction (PCR)-based molecular techniques have been useful for the genetic characterization of T. gondii . Serotyping methods based on polymorphic polypeptides have the potential to become the choice for typing T. gondii in humans and animals. In this review, we summarize conventional non-DNA-based diagnostic methods, and the DNA-based molecular techniques for the diagnosis and genetic characterization of T. gondii . These techniques have provided foundations for further development of more effective and accurate detection of T. gondii infection. These advances will contribute to an improved understanding of the epidemiology, prevention and control of toxoplasmosis.

Toxoplasma gondii is a parasite that infects a wide range of animals and causes zoonotic infections in humans. Although it normally only results in mild illness in healthy individuals, toxoplasmosis is a common opportunistic infection with high mortality in individuals who are immunocompromised, most commonly due to reactivation of infection in the central nervous system. In the acute phase of infection, interferon-dependent immune responses control rapid parasite expansion and mitigate acute disease symptoms. However, after dissemination the parasite differentiates into semi-dormant cysts that form within muscle cells and neurons, where they persist for life in the infected host. Control of infection in the central nervous system, a compartment of immune privilege, relies on modified immune responses that aim to balance infection control while limiting potential damage due to inflammation. In response to the activation of interferon-mediated pathways, the parasite deploys an array of effector proteins to escape immune clearance and ensure latent survival. Although these pathways are best studied in the laboratory mouse, emerging evidence points to unique mechanisms of control in human toxoplasmosis. In this Review, we explore some of these recent findings that extend our understanding for proliferation, establishment and control of toxoplasmosis in humans.Toxoplasmosis caused by the parasite Toxoplasma gondii is a common opportunistic infection with high mortality in individuals who are immunocompromised, most commonly due to reactivation of infection in the central nervous system (CNS). In this Review, Sibley and colleagues explore the interaction between host immune defences and parasite virulence factors with emphasis on bradyzoite differentiation and survival of T. gondii within the CNS.

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host.

Background The pathogenicity of Toxoplasma gondii is closely associated with its intracellular lytic cycle in host cells. Currently, the mechanisms by which T. gondii completes the lytic cycle remain unclear. The proline metabolism has been reported to be crucial for intracellular growth of pathogens by providing energy and nutrients. However, it remains unclear whether the intracellular growth and pathogenicity of T. gondii are related to proline metabolism. Methods The gene-edited strains of proline dehydrogenase ( Tgprodh ) were constructed by using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) technology. The effects of the Tgprodh gene on the growth in vitro and pathogenicity in vivo of the tachyzoites for T. gondii were studied through proliferation, plaque, invasion, egress and virulence assays. The effects of the Tgprodh gene on mitochondrial function were studied by using reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψm), adenosine triphosphate (ATP) assay kits, mitochondrial DNA (mtDNA) copy numbers, transmission electron microscopy (TEM) analysis, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The effects of the Tgprodh gene on proline metabolism were studied by using l -proline (L-Pro), l -glutamic acid (L-Glu), l -glutamine (L-Gln) assay kits, and RT-qPCR. Results TgPRODH, the first rate-limiting enzyme in proline metabolism, was identified to be encoded by T. gondii and localized in the cytoplasm of T. gondii . Deletion of the Tgprodh gene resulted in significant growth inhibition in vitro and reduced pathogenicity in vivo of T. gondii . Further study found that deletion of the Tgprodh gene caused damage to the mitochondrial morphology, decreased membrane potential, mtDNA copy numbers, and the production of ATP and ROS. The expression of genes for maintaining mitochondrial integrity was downregulated in the Tgprodh -knockout strain of T. gondii , while complementation of the Tgprodh gene restored these defects in this parasite. Meantime, the deletion of the Tgprodh gene resulted in the accumulation of proline, reduced the contents of glutamate and glutamine, and affected the expression of genes related to proline catabolism in T. gondii . Conclusions The present study found the significance of the Tgprodh gene for the intracellular growth and pathogenicity of T. gondii through regulating mitochondrial function and the proline metabolism and provided a novel insight to reveal intracellular survival strategies of T. gondii . Graphical Abstract

Toxoplasma is an obligate intracellular parasite that replicates in mammalian cells within a parasitophorous vacuole (PV) that does not fuse with any host organelles. One mechanism developed by the parasite for nutrient acquisition is the attraction of host organelles to the PV. Here, we examined the exploitation of host lipid droplets (LD), ubiquitous fat storage organelles, by Toxoplasma. We show that Toxoplasma replication is reduced in host cells that are depleted of LD, or impaired in TAG lipolysis or fatty acid catabolism. In infected cells, the number of host LD and the expression of host LD-associated genes (ADRP, DGAT2), progressively increase until the onset of parasite replication. Throughout infection, the PV are surrounded by host LD. Toxoplasma is capable of accessing lipids stored in host LD and incorporates these lipids into its own membranes and LD. Exogenous addition of oleic acid stimulates LD biogenesis in the host cell and results in the overaccumulation of neutral lipids in very large LD inside the parasite. To access LD-derived lipids, Toxoplasma intercepts and internalizes within the PV host LD, some of which remaining associated with Rab7, which become wrapped by an intravacuolar network of membranes (IVN). Mutant parasites impaired in IVN formation display diminished capacity of lipid uptake from host LD. Moreover, parasites lacking an IVN-localized phospholipase A2 are less proficient in salvaging lipids from host LD in the PV, suggesting a major contribution of the IVN for host LD processing in the PV and, thus lipid content release. Interestingly, gavage of parasites with lipids unveils, for the first time, the presence in Toxoplasma of endocytic-like structures containing lipidic material originating from the PV lumen. This study highlights the reliance of Toxoplasma on host LD for its intracellular development and the parasite's capability in scavenging neutral lipids from host LD.