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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.

Background The protozoan parasite Toxoplasma gondii has a worldwide distribution and a very wide host range, infecting most warm-blooded hosts. Approximately 30% of humanity is infected with T. gondii , but clinical toxoplasmosis is relatively infrequent. Toxoplasmosis has a wide range of clinical symptoms involving almost all organ systems. In most persons that acquire infection postnatally, symptoms (when present) are mild and mimic other diseases such as flu, Lyme disease, Q fever, hematological alterations, or mumps. It is likely that clinical disease is more common than reported. The ingestion of infected meat or food and water contaminated with oocysts are the two main modes of postnatal transmission of Toxoplasma gondii . The infective dose and the incubation period of T. gondii infection are unknown because there are no human volunteer experiments. Methods Here, I have critically reviewed outbreaks of clinical toxoplasmosis in humans for the past 55 years, 1966–2020. Information from oocyst-acquired versus meat-acquired infections was assessed separately. Results Most outbreaks were from Brazil. There were no apparent differences in types or severity of symptoms in meat- versus oocyst-acquired infections. Fever, cervical lymphadenopathy, myalgia, and fatigue were the most important symptoms, and these symptoms were not age-dependent. The incubation period was 7–30 days. A genetic predisposition to cause eye disease is suspected in the parasites responsible for three outbreaks (in Brazil, Canada, and India). Only a few T. gondii tissue cysts might suffice to cause infection, as indicated by outbreaks affecting some (but not all) individuals sharing a meal of infected meat. Conclusions Whether the high frequency of outbreaks of toxoplasmosis in humans in Brazil is related to environmental contamination, poor hygiene, socioeconomic conditions, or to genotypes of T. gondii needs investigation. Graphical Abstract

Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission.

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.

Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria.

Previous studies have reported high diversity between and within populations of Toxoplasma gondii in South America. In the present study, isolates of T. gondii from chickens were obtained from the Amazon region. Adult free-range chickens were acquired from 29 municipalities in the Brazilian Amazon region that included Acre (n = 9 municipalities), Amapá (n = 6), Amazonas (n = 6), Pará (n = 6), and Roraima (n = 2) states and from two municipalities in Peru, three in Bolivia, one in Guyana, and one in Venezuela. Heart, brain, and blood samples were collected from 401 chickens. Anti- T. gondii serum antibodies were detected in 273 (68.1%) chickens using the Modified Agglutination Test (MAT ≥ 5), and bioassays in mice were performed using 220 birds. Isolates were obtained from 116 (52.7%) chickens with antibody titers ≥ 20. Of these isolates, 93 (84.5%) led to acute sickness in more than 50% of the infected mice within 30 days post-inoculation. The 116 isolates were genotyped using multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-nPCR-RFLP) with 12 markers and 15 microsatellite (MS) markers. PCR-RFLP analysis revealed 42 genotypes from the 116 isolates. Of these, 20 (46.51%) genotypes are described for the first time. The most abundant genotype was ToxoDB PCR-RFLP genotype #7 with 40 isolates. A total of 83 genotypes were observed from the 116 isolates by MS analysis. The phylogenetic network constructed of T. gondii genotypes from current and previously reported isolates, using PCR-RFLP data, revealed five groups with clear indication of geographical separation of T. gondii population in the Amazon region versus the Southeastern region of Brazil. Such spatial diversity was also observed within the Amazon region. This study expands our knowledge of T. gondii population in South America and emphasizes the importance of genetic diversity and high mouse-virulence of the parasite in the Amazon region.

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.

Toxoplasma gondii is an intracellular protozoan whose intermediate hosts encompass nearly all warm-blooded animals, including humans and non-human primates (NHPs). In this study, molecular, serological, immunohistochemical and bioassay methods were used to investigate T. gondii infection in 17 captived NHPs in zoos from China between 2022 and 2023. The infection rate was higher in New World NHPs (83.3%) than in Old World NHPs (16.7%) ( P  = 0.2424). A viable strain of T. gondii was successfully isolated from the tissues of a black mangabey ( Lophocebus aterrimus ). The strain designated as TgMonkeyCHn3 was genotyped as ToxoDB #6, which had demonstrated virulence in Swiss Webster outbred mice. The ROP18/ROP5 allele combination was identified as 1/3. The emergence of this strain highlights the increased genetic diversity of T. gondii in East Asia and presents new challenges for the prevention of toxoplasmosis, particularly in captive NHPs. To the best of our knowledge, this is the first report of the ToxoDB genotype #6 isolated in East Asia and the black mangabey is a new host for T. gondii .

Toxoplasmosis is a major risk to chronically infected individuals, especially those who become immunocompromised. Although one-third of the globe is infected with Toxoplasma , no treatments prevent or eliminate cysts in part due to limited understanding of bradyzoite biology. The cyst is central to Toxoplasmosis, as transition from bradyzoites to tachyzoites drive pathology. In this study, we aim to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. Here, we discover ME49EW cysts from infected mice harbor multiple bradyzoite subtypes with distinct fates. Purified subtypes exhibit defined developmental pathways in animals and in primary astrocytes. Single-bradyzoite RNA-sequencing reveals five major subtypes within cysts. We further show that a crucial subtype in chronically infected mice is absent from a widely used in vitro model of bradyzoite development. Altogether, this work establishes new foundational principles of Toxoplasma cyst development and reactivation that operate during the intermediate life cycle of Toxoplasma . The authors investigate the heterogeneity of Toxoplasma bradyzoites prior to recrudescence. Tissue cysts from infected mice harbor multiple bradyzoite subtypes with distinct developmental fates as evidenced by single-bradyzoite RNAseq and FACS.

The development of simple, sensitive and rapid methods for the detection and identification of Toxoplasma gondii is important for the diagnosis and epidemiological studies of the zoonotic disease toxoplasmosis. In the past 2 decades, molecular methods based on a variety of genetic markers have been developed, each with its advantages and limitations. The application of these methods has generated invaluable information to enhance our understanding of the epidemiology, population genetics and phylogeny of T. gondii. However, since most studies focused solely on the detection but not genetic characterization of T. gondii, the information obtained was limited. In this review, we discuss some widely used molecular methods and propose an integrated approach for the detection and identification of T. gondii, in order to generate maximum information for epidemiological, population and phylogenetic studies of this key pathogen.