Feline Definitive Host Research Articles

The Zoonotic Aspect of Toxoplasma Gondii in Human and Animals: A Review

Toxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii, which may infect any nucleated cell in mammals. This parasite infects over two billion individuals worldwide, and while only a tiny fraction of infected persons may develop significant disease, the parasite's ubiquity makes it one of the most highly dangerous zoonotic infections in the world. This disease can cause serious encephalitis in immunocompromised persons, miscarriage or congenital malformations in the newborns if infected during pregnancy, and significant ocular illness even in immunocompetent people. The illness has a complicated aetiology, as it is caused by ingesting oocysts shed in the faeces of definitive feline hosts that pollute water. In this review we demonstrated the current insight into the zoonotic aspect of toxoplasmosis in humans, and animals providing a complete discussion of the environmental mechanisms via which T. gondii oocysts, and tissue cysts might infect animals and humans, such as contaminated foods, water, animals’ meat or soil. This review also included mode of transmission and preventative strategies outlining fundamental control measures for lowering the threat of oocyst direct contact, as well as chances for future collaboration and new studies possibilities targeting reducing oocysts load and tissue cyst-borne toxoplasmosis in people, farm and wild animals. Conclusively, the infection occurs in both humans and animals, causing abortion in women and serious economic losses in animals. Transmitted to humans horizontally by ingestion of food and water contaminated by oocysts, also meat of infected farm animals or vertically through the placenta. Cats get infected through ingestion of intermediate host-containing tissue cysts. Heating, salting, freezing and radiation are the main ways enforced to reduce tissue cyst burden in animal meat used for human consumption

AP2XII-1 is a negative regulator of merogony and presexual commitment in Toxoplasma gondii.

Sexual development is vital for the transmission, genetic hybridization, and population evolution of apicomplexan pathogens, which include several clinically relevant parasites, such as Plasmodium, Eimeria, and Toxoplasma gondii. Previous studies have demonstrated different morphological characteristics and division patterns between asexual and sexual stages of the parasites. However, the primary regulation is poorly understood. A transition from the asexual to the sexual stage is supposedly triggered/accompanied by rewiring of gene expression and controlled by transcription factors and chromatin modulators. Herein, we discovered a tachyzoite-specific transcriptional factor AP2XII-1, which represses the presexual development in the asexual tachyzoite stage of T. gondii. Conditional knockdown of AP2XII-1 perturbs tachyzoite proliferation by endodyogeny and drives a transition to a morphologically and transcriptionally distinct merozoite stage. The results also suggest a hierarchical transcriptional regulation of sexual development by AP2 factors and provide a path to culturing merozoites and controlling inter-host transmission of T. gondii.

High prevalence rates of Toxoplasma gondii in cat-hunted small mammals - Evidence for parasite induced behavioural manipulation in the natural environment?

Toxoplasma gondii causes one of the most frequent parasitic infections in vertebrates on earth. The present study aimed to assess the occurrence of T. gondii infection in cat-hunted wild small mammals, and to determine the circulating T. gondii genotypes in cat prey. There is evidence suggesting that T. gondii may manipulate rodents' behaviour enhancing transmission to their definitive feline host by facilitating predation. Given that most studies focusing on rodent behavior have been performed under laboratory conditions, we tested this hypothesis in the natural environment. We analysed 157 cat-hunted wild small mammals of six different species from Switzerland. Brain and skeletal muscle samples from each animal were tested for T. gondii DNA by PCR, and positive samples were genotyped using a multilocus sequence typing approach, including 10 genetic markers. Additionally, to evaluate exposure to cat faeces, the presence of Taenia taeniaeformis metacestodes was investigated at necropsy. The prevalence of T. gondii in cat-hunted Arvicola amphibius s.l. was 11.1% (7/63), 14.6% (7/48) in Apodemus spp., 13.6% (3/22) in Myodes glareolus, 6.7% (1/15) in Crocidura russula, and 0% in Microtus arvalis (0/8) and Sorex sp. (0/1). All completely genotyped T. gondii parasites, exhibited the ToxoDB #3 genotype, a Type II variant. We additionally analysed 48 trap-captured A. amphibius s.l., which all tested negative for T. gondii infection, contrasting with the higher prevalence in cat-hunted A. amphibius s.l. (0% vs. 11.1%; p=0.0176). Furthermore, T. taeniaeformis was detected in both groups, indicating widespread contamination with cat faeces in the sampled areas. These results provide evidence that T. gondii infected rodents are at higher risk to be predated by cats and therewith support the behaviour manipulation hypothesis.

Toxoplasma gondii infections are associated with costly boldness toward felids in a wild host

Toxoplasma gondii is hypothesized to manipulate the behavior of warm-blooded hosts to promote trophic transmission into the parasite’s definitive feline hosts. A key prediction of this hypothesis is that T. gondii infections of non-feline hosts are associated with costly behavior toward T. gondii’s definitive hosts; however, this effect has not been documented in any of the parasite’s diverse wild hosts during naturally occurring interactions with felines. Here, three decades of field observations reveal that T. gondii-infected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality. We discuss these results in light of 1) the possibility that hyena boldness represents an extended phenotype of the parasite, and 2) alternative scenarios in which T. gondii has not undergone selection to manipulate behavior in host hyenas. Both cases remain plausible and have important ramifications for T. gondii’s impacts on host behavior and fitness in the wild.

Gametocytes of Toxoplasma gondii in tissue culture

Toxoplasma gondii, included in the Phylum Apicomplexa, is a tissue parasite in the intermediate host and an intestinal parasite in the feline definitive host and needs no vector. In the tissues, it replicates asexually by endodyogeny, and endopolygeny; and asexually and sexually by schizogony and gametogony in cat ileum giving rise to infective oocysts. In vitro culture in cell lines provide a suitable model for studying proliferation of the parasite. Retrograde examination of permanent preparations demonstrating morphological forms of multiplying Toxoplasma in an in vitro culture cell line, revealed what may be considered gametogonic stages.

Acetylome analysis of the feline small intestine following Toxoplasma gondii infection.

Toxoplasma gondii is a protozoan parasite capable of infecting a large number of warm-blooded animals and causes serious health complications in immunocompromised patients. T. gondii infection of the feline small intestine is critical for the completion of the life cycle and transmission of T. gondii. Protein acetylation is an important posttranslational modification, which plays roles in the regulation of various cellular processes. Therefore, understanding of how T. gondii reprograms the protein acetylation status of feline definitive host can help to thwart the production and spread of T. gondii. Here, we used affinity enrichment and high-resolution liquid chromatography with tandem mass spectrometry to profile the alterations of the acetylome in cat small intestine 10 days after infection by T. gondii Prugniuad (Pru) strain. Our analysis showed that T. gondii induced significant changes in the acetylation of proteins in the cat intestine. We identified 2606 unique lysine acetylation sites in 1357 acetylated proteins. The levels of 334 acetylated peptides were downregulated, while the levels of 82 acetylated peptides were increased in the infected small intestine. The proteins with differentially acetylated peptides were particularly enriched in the bioenergetics-related processes, such as tricarboxylic acid cycle, oxidative phosphorylation, and oxidation-reduction. These results provide the first baseline of the global acetylome of feline small intestine following T. gondii infection and should facilitate further analysis of the role of acetylated protein in the pathogenesis of T. gondii infection in its definitive host.Electronic supplementary materialThe online version of this article (10.1007/s00436-020-06880-4) contains supplementary material, which is available to authorized users.

Infectious Causation of Abnormal Host Behavior: Toxoplasma gondii and Its Potential Association With Dopey Fox Syndrome.

The apicomplexan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, can infect all warm-blooded animals. T. gondii can subtly alter host behaviors—either through manipulation to enhance transmission to the feline definitive host or as a side-effect, or “constraint,” of infection. In humans, T. gondii infection, either alone or in association with other co-infecting neurotropic agents, has been reliably associated with both subtle behavioral changes and, in some cases, severe neuropsychiatric disorders, including schizophrenia. Research on the potential impact of T. gondii on the behavior of other long-lived naturally infected hosts is lacking. Recent studies reported a large number of wild red foxes exhibiting a range of aberrant behavioral traits, subsequently classified as Dopey Fox Syndrome (DFS). Here we assessed the potential association between T. gondii and/or other neurotropic agents with DFS. Live, captive foxes within welfare centers were serologically tested for T. gondii and, if they died naturally, PCR-tested for vulpine circovirus (FoxCV). Post-mortem pseudo-control wild foxes, obtained from pest management companies, were PCR-tested for T. gondii, FoxCV, canine distemper virus (CDV), canine adenovirus type (CAV)-1 and CAV-2. We also assessed, using non-invasive assays, whether T. gondii–infected foxes showed subtle behavioral alterations as observed among infected rodent (and other) hosts, including altered activity, risk, and stress levels. All foxes tested negative for CAV, CDV, CHV, and DogCV. DFS was found to be associated with singular T. gondii infection (captives vs. pseudo-controls, 33.3% (3/9) vs. 6.8% (5/74)) and singular FoxCV infection (66.7% (6/9) vs. 11.1% (1/9)) and with T. gondii/FoxCV co-infection (33.3% (3/9) vs. 11.1% (1/9)). Overall, a higher proportion of captive foxes had signs of neuroinflammation compared to pseudo-controls (66.7% (4/6) vs. 11.1% (1/9)). Consistent with behavioral changes seen in infected rodents, T. gondii–infected foxes displayed increased attraction toward feline odor (n=6 foxes). These preliminary results suggest that wild foxes with DFS are infected with T. gondii and likely co-infected with FoxCV and/or another co-infecting neurotropic agent. Our findings using this novel system have important implications for our understanding of both the impact of parasites on mammalian host behavior in general and, potentially, of the infectious causation of certain neuropsychiatric disorders.

Immediate Interferon Gamma Induction Determines Murine Host Compatibility Differences between Toxoplasma gondii and Neospora caninum.

Rodents are critical for the transmission of Toxoplasma gondii to the definitive feline host via predation, and this relationship has been extensively studied as a model for immune responses to parasites. Neospora caninum is a closely related coccidian parasite of ruminants and canines but is not naturally transmitted by rodents. We compared mouse innate immune responses to N. caninum and T. gondii and found marked differences in cytokine levels and parasite growth kinetics during the first 24 h postinfection (hpi). N. caninum-infected mice produced significantly higher levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) by as early as 4 hpi, but the level of IFN-γ was significantly lower or undetectable in T. gondii-infected mice during the first 24 hpi. "Immediate" IFN-γ and IL-12p40 production was not detected in MyD88-/- mice. However, unlike IL-12p40-/- and IFN-γ-/- mice, MyD88-/- mice survived N. caninum infections at the dose used in this study. Serial measures of parasite burden showed that MyD88-/- mice were more susceptible to N. caninum infections than wild-type (WT) mice, and control of parasite burdens correlated with a pulse of serum IFN-γ at 3 to 4 days postinfection in the absence of detectable IL-12. Immediate IFN-γ was partially dependent on the T. gondii mouse profilin receptor Toll-like receptor 11 (TLR11), but the ectopic expression of N. caninum profilin in T. gondii had no impact on early IFN-γ production or parasite proliferation. Our data indicate that T. gondii is capable of evading host detection during the first hours after infection, while N. caninum is not, and this is likely due to the early MyD88-dependent recognition of ligands other than profilin.

Immunization With a Live-Attenuated RH:ΔNPT1 Strain of Toxoplasma gondii Induces Strong Protective Immunity Against Toxoplasmosis in Mice.

Toxoplasmosis, one of the most important health-threatening diseases worldwide, is caused by Toxoplasma gondii, which infects a wide range of warm-blooded animals and humans, leading to enormous health and socioeconomic concerns. T. gondii can establish chronic infection to evade the immune response in hosts. Once a chronic infection has been established, the available treatments cannot efficiently control this stage of T. gondii efficiently. Moreover, the available treatments rely only on a few drugs, such as sulfapyridine and pyrimethamine, that tend to have severe side effects. Given these factors, vaccination has been considered to be the most efficient method to prevent and control this disease. However, there is currently lack of effective vaccine available for use to prevent toxoplasmosis apart form Toxovax®, the only available vaccine, which is used in sheep to prevent abortion. To address this problem, we knocked out the NPT1 gene of the type I T. gondii strain using the CRISPR-Cas9 system, constructed a live-attenuated vaccine and evaluated its protective efficacy in a mouse model. Immunization of mice with RH:ΔNPT1 induced a high level of Toxoplasma-specific IgG1, IgG2a and total IgG 42 days after immunization. There was a significant increase in the levels of cytokines in the splenocyte suspensions of RH:ΔNPT1-infected mice, and a mixed Th1/Th2 response was induced in the mice. Remarkably, after heterologous challenges with tachyzoites of the RH, PYS and Pru strains and cysts of the Pru strain by different infection routes, the immunized animals were protected from toxoplasmosis with a 100% survival rate, in both acute and chronic infection. In addition, compared with control mice, the Pru cyst load was clearly reduced in the brains of RH:ΔNPT1-infected immunization-mice. Our study demonstrated that the RH:ΔNPT1 strain was able to evoke strong anti-Toxoplasma immune responses and provide effective protection against parasite strains with different levels of virulence, suggesting that the RH:ΔNPT1 strain may represent a promising live-attenuated vaccine against toxoplasmosis, which is worthy of further evaluation in food-producing animals and in definitive feline host.

Risk factors and prevalence of antibodies for Toxoplasma gondii in diaphragmatic fluid in wolverines (Gulo gulo) from the Northwest Territories, Canada

Toxoplasma gondii, a zoonotic food borne parasite that can infect almost all warm-blooded animals including people, and ranks 4th among 24 most significant global foodborne parasites listed by the World Health Organization/United Nations Food and Agriculture Organization (FAO/WHO, 2014). Exposure to T. gondii has been reported in wildlife and people in the Canadian North, despite low densities of feline definitive hosts. The ecology of this host-parasite system could be affected by changing climate and landscape in boreal and sub-Arctic regions, and surveillance data are critically needed. Wolverines are an economically and culturally important species in northern Canada due to their valuable fur. Fluid obtained from diaphragmatic muscle of 127 wolverines (Gulo gulo) were tested for antibodies to T. gondii using an enzyme linked immunosorbent assay (ELISA). A seroprevalence of 62% (Confidence Interval (CI): 53–71%) was observed. This result indicates high levels of exposure, likely either through environmental contamination with T. gondii oocysts shed by infected wild felids, or consumption of carcasses/offal of other intermediate hosts containing tissue cysts with bradyzoites in tissues. We examined factors associated with seropositivity, including age, sex, harvest location, harvest location with respect to treeline, and body condition index. Adult (≥2 years) wolverines had 5.2 times higher odds of being sero-positive than juvenile (<1 years) wolverines. The highest seroprevalence was observed in wolverines from Sahtu and South Slave regions. Proportion of sero-positive wolverines harvested above and below the tree line was not significantly different (60% vs 65%). Age was the only significant predictor of T. gondii exposure in wolverines (using logistic regression analysis); further studies should target larger sample sizes. This study is an example of how fluid from diaphragmatic muscle can be used for screening for T. gondii antibodies in wolverines. The diaphragm, commonly collected for screening for another food borne parasite, Trichinella, in wildlife harvested for human consumption, can be used for screening of T. gondii exposure in wildlife. Due to their predatory and scavenging lifestyle and high trophic level, wolverines could serve as a sentinel species for T. gondii.

Serological and molecular detection of Toxoplasma gondii in terrestrial and marine wildlife harvested for food in Nunavik, Canada

BackgroundToxoplasma gondii, a zoonotic protozoan parasite, infects mammals and birds worldwide. Infection in humans is often asymptomatic, though illnesses can occur in immunocompromised hosts and the fetuses of susceptible women infected during pregnancy. In Nunavik, Canada, 60% of the Inuit population has measurable antibodies against T. gondii. Handling and consumption of wildlife have been identified as risk factors for exposure. Serological evidence of exposure has been reported for wildlife in Nunavik; however, T. gondii has not been detected in wildlife tissues commonly consumed by Inuit.MethodsWe used a magnetic capture DNA extraction and real-time PCR protocol to extract and amplify T. gondii DNA from large quantities of tissues (up to 100 g) of 441 individual animals in Nunavik: 166 ptarmigan (Lagopus lagopus), 156 geese (Branta canadensis and Chen caerulescens), 61 ringed seals (Pusa hispida), 31 caribou (Rangifer tarandus) and 27 walruses (Odobenus rosmarus).ResultsDNA from T. gondii was detected in 9% (95% CI: 3–15%) of geese from four communities in western and southern Nunavik, but DNA was not detected in other wildlife species including 20% (95% CI: 12–31%) of ringed seals and 26% (95% CI: 14–43%) of caribou positive on a commercial modified agglutination test (MAT) using thawed heart muscle juice. In geese, tissue parasite burden was highest in heart, followed by brain, breast muscle, liver and gizzard. Serological results did not correlate well with tissue infection status for any wildlife species.ConclusionsTo our knowledge, this is the first report on the detection, quantification, and characterization of DNA of T. gondii (clonal lineage II in one goose) from wildlife harvested for food in Nunavik, which supports the hypothesis that migratory geese can carry T. gondii into Nunavik where feline definitive hosts are rare. This study suggests that direct detection methods may be useful for detection of T. gondii in wildlife harvested for human consumption and provides data needed for a quantitative exposure assessment that will determine the risk of T. gondii exposure for Inuit who harvest and consume geese in Nunavik.

Kindness to the Final Host and Vice Versa: A Trend for Parasites Providing Easy Prey?

Traditionally the 'extended phenotype' concept refers to parasites that manipulate host phenotype to increase parasite fitness. This includes parasites who render intermediate hosts more susceptible to predation by final hosts. We explore here the proposition that an evolutionary driver in such cases is the energetic benefit to the final host, in addition to increased parasite fitness. We will review some well established host-manipulation models, where such a scenario seems likely. One example is provided by the protozoan Toxoplasma gondii, which conspicuously impairs predator avoidance in rodents. Pathologies in humans that acquire T. gondii are known, but infection in adult feline definitive hosts are most commonly asymptomatic and apparently innocuous. In another well documented case of parasite-mediated trophic transmission, trematode (Euhaplorchis californiensis) infected killifish (Fundulus parvipinnis) abandon normal caution and exhibit a range of behaviors which makes them more conspicuous to predatory birds. The birds get a free meal, but the presence of adult trematodes in the gut would seem to incur few if any negative consequences for the birds. There are exceptions to this pattern also among cases of parasite mediated trophic transmission, but major pathology in definitive hosts seems for the most part restricted to cases where manipulated intermediate hosts are of minor energetic importance. Current theories for the evolution of reduced pathogenicity in predatory final hots primarily focus on parasites minimizing pathogenicity to increase their own reproductive output and/or avoid selection on host preference for non-infected prey types. Here we advocate another alternative: If or when the benefit of increased prey acquisition outweighs pathogenicity or resource drain, there should be little or no selection on final hosts to minimize parasite infections. This mean that not only will host avoidance of infection not develop, but the molecular arms race for increased immunological defense will also likely be halted in such cases.

Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii

BackgroundAlthough sexual reproduction of the parasite Toxoplasma gondii exclusively occurs in the cat intestine, knowledge about the alteration of gene expression in the intestine of cats infected with T. gondii is still limited. Here, we investigated the temporal transcriptional changes that occur in the cat intestine during T. gondii infection.MethodsCats were infected with 100 T. gondii cysts and their intestines were collected at 6, 12, 18, 24, 72 and 96 hours post-infection (hpi). RNA sequencing (RNA-Seq) Illumina technology was used to gain insight into the spectrum of genes that are differentially expressed due to infection. Quantitative RT-PCR (qRT-PCR) was also used to validate the level of expression of a set of differentially expressed genes (DEGs) obtained by sequencing.ResultsOur transcriptome analysis revealed 2363 DEGs that were clustered into six unique patterns of gene expression across all the time points after infection. Our analysis revealed 56, 184, 404, 508, 400 and 811 DEGs in infected intestines compared to uninfected controls at 6, 12, 18, 24, 72 and 96 hpi, respectively. RNA-Seq results were confirmed by qRT-PCR. DEGs were mainly enriched in catalytic activity and metabolic process based on gene ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that transcriptional changes in the intestine of infected cats evolve over the course of infection, and the largest difference in the enriched pathways was observed at 96 hpi. The anti-T. gondii defense response of the feline host was mediated by Major Histocompatibility Complex class I, proteasomes, heat-shock proteins and fatty acid binding proteins.ConclusionsThis study revealed novel host factors, which may be critical for the successful establishment of an intracellular niche during T. gondii infection in the definitive feline host.

Insights into the molecular basis of host behaviour manipulation by Toxoplasma gondii infection.

Typically illustrating the 'manipulation hypothesis', Toxoplasma gondii is widely known to trigger sustainable behavioural changes during chronic infection of intermediate hosts to enhance transmission to its feline definitive hosts, ensuring survival and dissemination. During the chronic stage of infection in rodents, a variety of neurological dysfunctions have been unravelled and correlated with the loss of cat fear, among other phenotypic impacts. However, the underlying neurological alteration(s) driving these behavioural modifications is only partially understood, which makes it difficult to draw more than a correlation between T. gondii infection and changes in brain homeostasis. Moreover, it is barely known which among the brain regions governing fear and stress responses are preferentially affected during T. gondii infection. Studies aiming at an in-depth dissection of underlying molecular mechanisms occurring at the host and parasite levels will be discussed in this review. Addressing this reminiscent topic in the light of recent technical progress and new discoveries regarding fear response, olfaction and neuromodulator mechanisms could contribute to a better understanding of this complex host-parasite interaction.

Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii.

ABSTRACTThe Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated ovarian tumor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites exhibited a fitness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting deviations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregulation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the attainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The resulting dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of specific cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms underlying the plasticity of apicomplexan cell cycle architecture.

The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs.

Toxoplasma gondii has a complex life cycle characterized by multiple differentiation steps that are essential for its survival in both human and definitive feline host. Several studies have demonstrated the importance of phosphorylations by protein kinases during the life cycle of T. gondii. However, very little is known about protein phosphatases and their regulators in the parasite. We report the molecular and functional characterization of the T. gondii ortholog of the inhibitor-2 protein, designated TgI2. We show that TgI2 encompasses conserved motifs involved in the interaction and modulation of the phosphatase activity of T. gondii protein phosphatase 1, named TgPP1. We show that a specific combination of motifs is involved in binding and/or inhibition of the TgPP1 activity. We show here that the TgI2 protein is a potent inhibitor of TgPP1 phosphatase activity. TgI2 SILK and RVxF motifs are critical for regulating the activity of TgPP1, a feature that is common with the higher eukaryotes inhibitor-2 protein.

Purification Toxoplasma gondii Tissue Cysts Using Percoll Gradients.

The protozoan parasite Toxoplasma gondii is capable of infecting all warm-blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the central nervous system and muscle during the asexual cycle within all chronically infected warm-blooded hosts. These tissue cysts are populated with slow-growing bradyzoites, which until recently have been thought to be dormant entities in the context of immune sufficiency. Reactivation to active growth during immune suppression is of critical clinical importance. However, little is known about tissue cysts or the bradyzoites they house, as the diversity of tissue cysts cannot be replicated in cell culture systems. This protocol for optimization of tissue cyst purification from the brains of infected mice using Percoll gradients provides an efficient means to recover in vivo-derived tissue cysts that can be applied to imaging, cell biological, biochemical, transcriptomic, and proteomic analyses. © 2017 by John Wiley & Sons, Inc.

An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells.

Toxoplasmosis is a zoonotic infection affecting approximately 30% of the world’s human population. After sexual reproduction in the definitive feline host, Toxoplasma oocysts, each containing 8 sporozoites, are shed into the environment where they can go on to infect humans and other warm-blooded intermediate hosts. Here, we use an in vitro model to assess host transcriptomic changes that occur in the earliest stages of such infections. We show that infection of rat intestinal epithelial cells with mature sporozoites primarily results in higher expression of genes associated with Tumor Necrosis Factor alpha (TNFα) signaling via NF-κB. Furthermore, we find that, consistent with their biology, these mature, invaded sporozoites display a transcriptome intermediate between the previously reported day 10 oocysts and that of their tachyzoite counterparts. Thus, this study uncovers novel host and pathogen factors that may be critical for the establishment of a successful intracellular niche following sporozoite-initiated infection.

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat.

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

Seroprevalence, isolation and co-infection of multiple Toxoplasma gondii strains in individual bobcats (Lynx rufus) from Mississippi, USA

Toxoplasma gondii causes lifelong chronic infection in both feline definitive hosts and intermediate hosts. Multiple exposures to the parasite are likely to occur in nature due to high environmental contamination. Here, we present data of high seroprevalence and multiple T. gondii strain co-infections in individual bobcats (Lynx rufus). Unfrozen samples (blood, heart, tongue and faeces) were collected from 35 free ranging wild bobcats from Mississippi, USA. Toxoplasma gondii antibodies were detected in serum by the modified agglutination test (1:≥200) in all 35 bobcats. Hearts from all bobcats were bioassayed in mice and viable T. gondii was isolated from 21; these strains were further propagated in cell culture. Additionally, DNA was extracted from digests of tongues and hearts of all 35 bobcats; T. gondii DNA was detected in tissues of all 35 bobcats. Genetic characterisation of DNA from cell culture-derived isolates was performed by multiplex PCR using 10 PCR-RFLP markers. Results showed that ToxoDB genotype #5 predominated (in 18 isolates) with a few other types (#24 in two isolates, and #2 in one isolate). PCR-DNA sequencing at two polymorphic markers, GRA6 and GRA7, detected multiple recombinant strains co-infecting the tissues of bobcats; most possessing Type II alleles at GRA7 versus Type X (HG-12) alleles at GRA6. Our results suggest that individual bobcats have been exposed to more than one parasite strain during their life time.