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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.

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.

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.

Toxoplasmosis is a food- and waterborne zoonosis of a great importance, ranked as the fourth most important foodborne parasitosis in the world. The objective of this study was to determine the seroprevalence and molecular prevalence of Toxoplasma gondii in wild boar ( Sus scrofa ) and to identify genotypes circulating in Croatia. A total of 103 wild boars from four hunting areas in Croatia were screened. Cardiac fluid samples were tested for anti- T. gondii antibodies by MAT, while heart samples were tested for T. gondii DNA using qPCR. The seroprevalence in the cardiac fluid reached 54.4%, while 19.4% of the heart samples were positive by qPCR. The highest seroprevalence was detected in the hunting ground Visocica with 95.6%. Partial microsatellite genotyping was achieved for 2/20 qPCR-positive heart samples suggesting type II strain of T. gondii . Relatively high seroprevalence and detection of T. gondii DNA in tested samples highlights the risk of human infection through consumption of undercooked meat. Further studies focusing on parasite detection in wild boar meat, especially across different climates, are needed to understand regional differences. Expanding research to other wildlife species is also important to clarify their role in the spread of T. gondii .

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.

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 .

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.

Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission. Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7). A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.