Effects of latent infection of Toxoplasma gondii strains with different genotypes on mouse behavior and brain transcripts

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In China, the two different isolates, Chinese I ( ToxoDB#9 ) and Chinese III are dominant epidemic strains widely spreading in humans and domestic animals and can lead to latent infection in host brain tissues, but the comparison of their manipulation patterns and mechanisms remains unclear. Methods Tachyzoites of TgWh6 (Wh6) strain and TgCtLHG (LHG) strain were used for establishing in vitro infection models within mouse microglia BV2 cells, and the differences in their invasion and proliferation patterns were observed. C57BL/6J mice were used to establish in vivo latent infection models. After behavioral tests, the differential expressed transcripts (DETs) of the infected and control animals' cerebral cortex were sequenced by Nanopore RNA-seq. Functional differences of DETs were analyzed by Gene Ontology enrichment analysis (GO), Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG), and protein-protein interaction (PPI) and cluster analysis. Expression of the key candidates were verified by quantitative polymerase chain reaction (qPCR). Results In our infection models, we found that Wh6 had more vigorous invasion and proliferation abilities in vitro , while LHG had greater ability to form cysts in vivo . In the latent infection phase, behavioral changes including spatial working memory, cognitive and motor abilities, and anxiety were apparently observed in both Wh6 and LHG infected mice, however, the LHG group showed more serious anxiety. Among DETs, genes related to MHC class II molecules were significantly up-regulated both in the infected mice, while genes related to synaptic transmission and neurodegenerative diseases were respectively down-regulated in the infected groups. The downregulated DETs of Sept4 , Kcng4 , Unc13c , and Prkcg in the WH6 group, which are related to synaptic transmission; and Ndrg2 and Arc in the LHG group, which are related to neurodegenerative diseases, would be selected to be the key candidates in the latent infection phase. Conclusion Compared with WH6, although LHG has a milder invasion ability, it can cause increased behavioral disorders in hosts. Genes related to synaptic transmission and neurodegenerative diseases may be the main causes of host mental and behavioral disorders. Toxoplasma gondii Cerebral cysts Differentially expressed transcripts Nanopore RNA-seq Mental and behavioral disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background T. gondii is an intracellular apicomplexan parasite infecting nearly all warm-blooded animals worldwide [1, 2]. About 30–50% of the global population has been exposed to or infected with T. gondii [3]. T. gondii is highly prevalent in Western Europe, South America, and Africa [4, 5], with infection rates ranging from 10% in the United States to > 50% in France, Colombia, and Brazil [6, 7, 8]. Moreover, a 2018 survey on toxoplasmosis in China demonstrated that the T. gondii infection rate among the Chinese population was 8.2%, and 23.7% among livestock [9]. Because of its high prevalence and widespread distribution in its intermediate hosts including livestock, poultry, and pets, T. gondii is increasingly recognized as a considerable risk to human health and the livestock industry. Tachyzoites could rapidly invade host's blood and various organs including eyes and internal organs, resulting in serious acute infections, particularly in immunodeficient patients. For instance, toxoplasmosis-induced encephalitis is a common cause of death in patients with Acquired Immunodeficiency Syndrome (AIDS) [10]. Although the clinical symptoms in infected individuals with normal immune function are not apparent in the acute phase, tachyzoites could also transform into bradyzoites and tissue cysts in the latent phase. Notably, cerebral cysts, the most common T. gondii -associated tissue cyst type, could persistently infect brain tissues, leading to mental and behavioral disorders [11, 12]. In rodent infection models, it can trigger various behavioral disorders including loss of natural fear, an increased desire to explore, and a decline in cognitive function [13, 14, 15]. Further, it has been proved that neuroinflammation and synaptic alterations were associated with the behavioral abnormal post-chronic infections with T. gondii strains, for example, the synaptic proteins EAAT2 and GABAAα1, which are involved in excitation/inhibition balance in Central Nervous System (CNS), could be down-regulated after infection, and accompanied with increased microglia activation [16, 17]. In some cases, different phenotypes such as depression, anxiety, and reduced motor capacity have also been reported [18, 19, 20]. So far, there are already more than 300 publications have shown that various types of psychiatric disorders, ranging from schizophrenia to depression, suicidal tendencies, Alzheimer's disease, road rage-induced traffic accidents, and even positive behaviors (e.g., entrepreneurship and risk-taking) are significantly associated with T. gondii infection [21, 22]. However, the mental behavioral manipulation patterns caused by different T. gondii strains with different genotypes, as well as the different pathogenic characteristics, particularly for Chinese T. gondii isolates, still remain unclear [13, 14, 23]. Up to now, there are 12 genotypes of Chinese T. gondii strains have been identified, and the dominant strain Chinese I ( ToxoDB#9 ) occupied 66.36% of all genotypes, followed by type I and II (variants) [24, 25, 26]. In particular, the Chinese III strain, also widespread in humans and domestic animals, is phylogenetically close to the typical type I virulent strains such as RH and GT1 [27] Although its virulence is weaker, it is more likely to form brain cysts than Chinese I isolates [28, 29]. However, there are neither study has reported the phenotype of behaviors of hosts infected with Chinese III strains, nor has compared the manipulation patterns and mechanisms of different Chinese T. gondii isolates. Therefore, in the present study, we have established both in vitro and in vivo infection models with TgCtwh6 (Wh6; feline-sourced Ⅰ×Ⅱ hybrid strain) and TgCtLHG (LHG; human-sourced type III strain), respectively. Based on these, we have analyzed the differences in host symptoms in both acute and latent infection phases and assessed the behavioral differences as well as the full-length cerebral cortex transcript expression before and after chronic infection. Our findings may provide a basis for an in-depth interpretation of parasite-mediated mental and behavioral manipulation mechanisms with different genotypes. Methods Animals and parasites TgCtWh6 (Wh6, a Chinese Ⅰ×Ⅱ hybrid genotype strain, isolated from a stray cat in Wuhan, China) and TgCtLHG (LHG, a type III genotype strain, isolated from the peripheral blood of a patient with toxoplasmosis) were kindly provided by Prof. Jilong Shen at Key Laboratory of Microbiology and Parasitology, Anhui Medical University (Hefei, Anhui, China). Brain cysts of Wh6 and LHG were continuously transmitted to the animal hosts BALB/c mice via cyst gavage [30]. Specific pathogen-free C57BL/6J mice, aged 6–8 weeks, were purchased from Pengyue Experimental Animal Breeding (Jinan, Shandong, China; animal license number: SCXK(Lu) 20220006). All animals were maintained under a constant temperature (22°C ± 2°C) and humidity (55% ± 5%) under a 12 h day-night cycle, with free access to food and water. The drinking water was sterilized through high-pressure disinfection, and the feed and bedding were sterilized using ultraviolet light. Cell culture and in vitro infection BV2 cells were routinely cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, USA) at 37°C in a humidified atmosphere of 5% CO 2 . When the passage density was reached, all cells were digested with 0.25% EDTA-trypsin, divided into flasks at a 1:2 ratio, and cultured at 37°C under 5% CO 2 until 70–80% of the cells fused. Next, the medium was replaced with fresh high-glucose DMEM containing 3% fetal bovine serum. After 12 h, the cells were used in an in vitro experiment with T. gondii tachyzoites. In total, six-cell culture dishes (35 mm × 10 mm) were placed in sterile conditions and numbered, and 2.4 × 10 5 cells were inoculated into each dish. After 12 h of culture at 37°C under 5% CO 2 , the medium was replaced, and the cells were allowed to grow to 85% confluency. Tachyzoites were co-cultured with BV2 cells at a 1:2 ratio, and the infected cells were observed for invasion status at 12, 24, 48, and 72 h after infection. Construction of chronic infection mouse model In total, 60 female and 60 male C57BL/6J mice, aged 6–8 weeks, were used to establish chronic infection models. Animals were divided into three groups: control group (Con), Wh6-infected group (TgW), and LHG-infected group (TgL). Each group included 20 female and 20 male mice. Each infection group was gavaged with 0.2 mL of cerebral tissue suspension containing 10 cysts, and the control group was gavaged with 0.2 mL of saline. After 45 days of inoculation, one mouse from each group was randomly selected and euthanized to obtain brain tissue. Besides, cysts were observed under a microscope to determine infection effectiveness. Clinical observation We observed clinical symptoms exhibited by tachyzoite-infected mice daily over 0–42 days after infection. For each infected mouse, the onset and duration of acute symptoms, such as back arching, erect hair, tremors, limb weakness, and eye disturbances, were recorded. The sex, survival time, and number of dead mice were also documented. Behavioral testing Y-maze test (YM) Each mouse was placed in the starting arm of a Y-maze and allowed to explore the three arms. The exploration behavior of a test mouse was recorded by a camera; in particular, we recorded the total distance traveled, the number of entries into each arm, and spontaneous alternation [= (total number of alternations/number of maximum alternations) × 100%]. Open-field test (OFT) An OFT was conducted using a camera to detect the movements of animals in a 50 × 50 cm 2 arena in a quiet, soundproof, dimly lit (≈ 20 lx) room. A mouse was placed in the center of the arena and allowed to move freely for 10 min. The movements of each mouse in each of the three groups were recorded using an automatic video tracking system for 1 min. In particular, we recorded the total distance traveled, average speed, immobility time, number of entries into the central area, distance traveled within the central area, and time spent in the central area. Predator Odor-Induced OFT This test was used to evaluate the host's fear response to predators. The method was similar to the OFT, but the urine of cat was placed sequentially in one corner of each compartment to assess how much time the mice spent in the urine-exposed areas. Brain tissue collection and T. gondii cysts counting After 45 days of infection, the mice were anesthetized, euthanized, and immediately dissected to remove the whole brain. The brain tissue was washed with sterile phosphate-buffered saline (PBS) and placed in a Keeper preservation solution. A random sample of mice from the infected groups was euthanized under sterile conditions with ether anesthesia, and the whole brain tissue was segmented along the longitudinal axis into left and right hemispheres, with the cerebral cortex, hippocampus, and olfactory bulb separated. Each part was thoroughly homogenized in 2 mL of PBS. Next, 10 µL of the homogenate was used to prepare a smear. The number of T. gondii cysts was counted under a microscope (400× magnification), and for each mouse, an average value was calculated from three smears. RNA extraction RNA extraction After completion of all behavior tests, the whole brains of all tested mice were excised and immediately frozen in liquid nitrogen. Since cysts are predominantly localized in cortical areas of the brain [31], the whole cortical regions of a tested mouse were separated and used to be an RNA sample. The total RNA in each sample was extracted using TRIzol reagent (Thermo Fisher, USA), according to the manufacture's instructions. The total absorbance (A = A 260 /A 280 + A 260 /A 230 ) was detected to analyze the purity of RNA, and the Aglient 2100 Bioanalyzer was used to detect RNA concentration and integrity. Library construction and Nanopore RNA sequencing We used Oxford Nanopore Technologies-based single-molecule real-time sequencing technology (i.e., ONT RNA-seq) for sequencing full-length RNA transcripts. Total RNA was extracted from mouse brain tissue, and mRNA was obtained through magnetic bead enrichment. The library was subsequently constructed, and the high-throughput sequencing was performed by Biomarker Technologies Co., LTD. The raw sequence was evaluated for quality and filtered to remove adapter sequences, short segments, and low-quality sequences; finally, high-quality read lengths were analyzed, and data on differential representative transcripts were obtained. Functional enrichment analysis Differentially expressed transcriptome data were obtained through quantitative analysis of transcriptome data from the brains of infected and control mice. The resulting sequences of differently expressed transcripts were compared with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes libraries (KEGG) to obtain relevant functional annotations and differentially expressed transcripts (DETs). To understand the changes in transcription levels in infected mice, we analyzed DETs with the following conditions: fold change (i.e., fold differences in DETs expression) is ≥ 1.5, and P (i.e., significance screening index for DETs) is < 0.05. The classification and enrichment of DETs were performed to GO functional annotation and KEGG pathway analyses in order to select the top 10 up-regulated and top 10 down-regulated DETs. The P values for these analyses were calculated using edge R, and P ≤ 0.05 was considered to indicate statistically significant enrichment. Protein-protein interaction and cluster analysis The potential interactions between the encoded proteins were retrieved from the string database, the protein-protein interaction (PPI) networks were constructed, and the clustering analysis of protein populations was performed using clustering options [32]. Statistics analysis All data, expressed as means ± standard errors of the means (SEMs), were analyzed using GraphPad Prism (version 8.0). We used the Shapiro-Wilk normality test followed by Student's t-test to compare the data from the two groups. Moreover, One-way ANOVA analysis followed by the post hoc Turkey test was used to analyze the effects of T. gondii infection. P < 0.05 was considered to indicate statistical significance. Results Wh6 tachyzoites are more invasive and proliferative than LHG tachyzoites within glial cells in vitro By the continuous detection under the inverted microscope, most tachyzoites of either strain could adhere to BV2 cell surfaces at 12 h of co-culturing, with only a few of them entering the cytoplasm. Almost all of the tachyzoites could complete cell invasion at 24 h after infection. At 48 h of co-culturing, the parasites began to divide, some Wh6-infected BV2 cells demonstrated a protrusion filled with the parasites on their membranes. By 72 h after infection, many parasites were released and began to start a new round of invasion (Fig. 1 a, upward arrow). However, the tachyzoites of LHG could not lead to cell membrane protrusion or parasite release until 72 h after co-culturing (Fig. 1 a, downward arrow). Thus, our results showed that Wh6 has stronger invasive and proliferative ability in BV2 cells than LHG. The infection symptoms caused by Wh6 and LHG tachyzoites, and the number of brain cysts were different At 5 days after infection, the infected mice began to exhibit acute symptoms such as hunched back, piloerection, tremor, limb weakness, eye discoloration, and decreased activity (Fig. 1 b). This acute onset phase cloud lasts for 20 days after infection. In general, TgW mice tended to have more severe eye damage, while TgL mice were more likely to demonstrate hind limb weakness (Table 1 ). We found that all infected mice could demonstrate cyst formation in brain tissues at 21 days after infection (Fig. 1 b), but the number of brain cysts in the TgL group was significantly more than those in the TgW group (Fig. 1 c). Surprisingly, we also found that all of the dead mice were female, and most of them belonged to the TgL group (Table 1 ). As a result, we speculated that LHG strain may have a sex bias for host fatality. Table 1 Continuous symptom observations of mice in TgW and TgL groups (n = 20) Mouse sex T. gondii strain Survival (days) Death rate a (%) Acute symptom rate (%) Hogback Piloerection Shiver Hind leg weakening Whitening of eyes Male Wh6 / 0 (0/20) 100 (20/20) 50 (11/20) 100 (20/20) 0 (0/20) 12.5 (2/20) LHG / 0 (0/20) 100 (20/20) 50 (12/20) 100 (20/20) 100 (20/20) 0 (0/20) Female Wh6 16 10.0 (2/20) 100 (20/20) 87.5 (18/20) 100 (20/20) 0 (0/20) 0 (1/20) LHG 12 65.0 (13/20) 100 (20/20) 100 (20/20) 100 (20/20) 100 (20/20) 0 (0/20) a Death rate = (number of deaths / number of infected individuals × 100%). Different genotypes of T. gondii strain could cause different types of mental and behavioral disorders The Y-maze results showed that chronic T. gondii infection impaired the spatial working memory ability of mice. Although the total distance traveled in the YM test did not differ significantly among the TgW, TgL, and Con groups (Fig. 2 a), the autonomous alternation rate decreased significantly in both infected groups compared to the Con group ( P < 0.01; Fig. 2 b), indicating that both WH6 and LHG could impair the spatial working and short-term memory ability of the hosts in the chronic infection phase, with a similar degree. A representative motion trajectory diagram is shown in Fig. 2 c. The OFT results showed that the TgL group had more severe motor impairments and higher anxiety levels. Compared with the Con group, both TgW and TgL groups exhibited significantly lower average speeds and longer immobility times, however, the TgW group showed significantly longer total distance traveled ( P < 0.05 or P < 0.001; Fig. 2 d-f), and an increase in the total number of entries into the central zone but a reduction in the total distance traveled and time spent in the central zone ( P < 0.01 or P < 0.001; Fig. 2 g-i). Thus, the chronic infection may cause slight motor impairments along with anxiety-like behavior in the hosts. Compared with TgW, the TgL group demonstrated a slightly shorter total distance ( P < 0.05), lower average speed ( P < 0.01), and significantly higher immobility time ( P < 0.001; Fig. 2 d–f), indicating that LHG strain had relatively more severe motor impairments, consistent with our results in the acute infection phase. In particular, mice in the TgL group showed fewer central zone entries but slightly longer time spent in the central zone ( P < 0.05 or P < 0.01; Fig. 2 g-i), suggesting that LHG strain may be more likely to cause hosts' anxiety. A representative motion trajectory diagram is shown in Fig. 2 j. The cat urine OFT results showed that both TgW and TgL mice had the loss of natural fear of predators, while TgW group was more significant. We collected urine samples from domestic cats and conducted a predator-induced mineshaft OFT. The results showed that all the infected mice spent significantly longer time in the urine-containing area than the control mice (Fig. 2 k), consistent with the previous results [31, 33]. However, mice in the TgW group appeared more significant fear loss degree ( P < 0.01) than those of the TgL group ( P < 0.05). A representative motion trajectory diagram is shown in Fig. 2 l. Identification of DETs among TgW, TgL, and Con groups The third-generation high-throughput ONT RNA-seq results demonstrated that the TgW and TgL groups included 1,789 and 1,119 DETs compared with the Con group, respectively. The TgW and TgL groups included 1,659 and 1,089 up-regulated DETs, respectively; and 130 and 30 down-regulated DETs, respectively. In the comparison of the TgW and TgL groups, 466 DETs were detected, of which 223 were up-regulated and 243 were down-regulated, respectively (Fig. 3 ). Chronic infection can intensively influence the immune response and synaptic transmission-related processes of the host Compared to the Con group, the top 10 up-regulated BPs in the TgW group were immune response, response to protists, cellular response to interferon β, antigen processing and presentation, inflammatory response, cellular response to interferon γ, innate immune response, defense response, defense response to viruses, and response to bacteria. The top 10 up-regulated KEGG pathways were Epstein–Barr (EB) virus infection, allogenic transplant rejection, antigen processing and presentation, phagosome, xenograft host defense, autoimmune thyroid disease, viral myocarditis, type I diabetes, leishmaniasis, and cell adhesion molecules (Fig. 4 a, left ) . The top 10 down-regulated BPs were the Gamma-aminobutyric acid (GABA) signaling pathway, protein homo-oligomerization, morphogenesis of the camera-shaped eye development, GABAergic synaptic transmission, 9-cis retinoic acid biosynthesis process, chloride ion transport, vesicle fusion-positive regulation, inhibitory synapse assembly, hair cycle regulation, and calcium-dependent exocytosis positive regulation. The top 10 down-regulated KEGG pathways were nicotine addiction, retrograde endogenous cannabinoid signaling, morphine addiction, GABAergic synaptic transmission, neuroactive ligand-receptor interactions, taste transduction, dopaminergic synapses, synaptic vesicle cycles, epidermal growth factor receptor tyrosine kinase inhibitor resistance, and other types of o-glycan biosynthesis ( Fig. 4 a, right). Taken together, these results demonstrated that infection with Wh6 mainly leads to up-regulation of processes involved in immune responses associated with pathogenic infection and down-regulation of processes related to synaptic signaling. Simultaneously, compared to the Con group, the top 10 up-regulated BPs in the TgL group were immune response, cellular response to interferon β, cellular response to interferon γ, antigen processing and presentation, response to protists, defense response, response to bacteria, innate immune response, defense response to gram-positive bacteria, and inflammatory response. The top 10 up-regulated KEGG pathways were EB virus infection, allogenic transplant rejection, phagosome, antigen processing and presentation, xenograft host defense, viral myocarditis, Staphylococcus aureus infection, type I diabetes, and leishmaniasis (Fig. 4 b, left). The top 10 down-regulated BPs were the regulation of platelet-derived growth factor generation, regulation of vascular endothelial growth factor generation, negative regulation of the extracellular signal-regulated kinase 1 and 2 cascades, negative regulation of cell growth, membrane invagination, synaptic vesicle cycle, vesicle-mediated transport in synapses, positive regulation of adipocyte differentiation, negative regulation of hepatocyte differentiation, and negative regulation of forebrain neuron differentiation. The top 10 down-regulated KEGG pathways were alcohol addiction, chemokine signaling pathways, human cytomegalovirus infection, HIV type I infection, GABAergic synapses, circadian rhythms, opioid addiction, serotonergic synapses, cholinergic synapses, and glutamatergic synapses (Fig. 4 b, right). Similarly, these results indicated that infection with LHG would also lead to the up-regulation of immune responses, and the down-regulation of synaptic signaling, as well as processes related to cell differentiation. LHG strain may cause enhanced variation of synaptic transmission and metabolic secretion Compared to the TgW group, the top 10 up-regulated BPs in the TgL group were the regulation of neuronal synaptic plasticity, response to nicotine, positive regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor activity, behavior, intracellular transport, cellular response to interleukin 8, positive regulation of synaptic transmission, regulation of calcium ion transport, long-term memory, and regulation of cation channel activity. The top 10 up-regulated KEGG pathways were aldosterone synthesis and secretion, glutamatergic synapses, calcium signaling pathways, pancreatic secretion, salivary secretion, gastric secretion, cosecretion and action of parathyroid hormones, retrograde endocannabinoid signaling, long-term potentiation, and cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathways (Fig. 4 c, left). The top 10 down-regulated BPs were the cellular response to gamma interferon, antigen processing, and presentation, positive regulation of type II hypersensitivity reactions, inflammatory responses, acetylcholine receptor signaling pathways, immune responses, lipoprotein lipid oxidation negative regulation, cellular response to interferon β, fibroblast proliferation negative regulation, and complement activation. The top 10 down-regulated KEGG pathways were phagosomes, plant-pathogen interactions, allograft rejection, type 1 diabetes, autoimmune thyroid disease, S. aureus infection, EB virus infection–viral myocarditis, and systemic lupus erythematosus (Fig. 4 c, right). These results demonstrated that the regulation of hosts' synaptic transmission and metabolic secretory function may be enhanced by LHG strain, yet the inflammatory immune responses showed more weakness in the TgL group. Verification of the key DET candidates DETs with high expression changes, top GO annotation, and top KEGG enrichment were selected to obtain heatmaps of gene expression profiles corresponding to the top 10 key candidate DETs (Fig. 5 a-c). Among them, the first four up-regulated key candidates were selected for qPCR verification in each group, and the results showed that most of the gene expression was consistent with our ONT RNA-seq results (Fig. 5 d and Additional file 1: Table S1 ). In particular, the mRNA expression of H2-Aa and Fcgr4 in the TgL group, H2-Q7 in the TgL group, and Arc and Itpr1 in the TgW vs TgL group were significantly up-regulated. Similarly, the verification of down-regulated key candidates showed that a majority of the selected gene expression results were consistent with the DETs results (Fig. 5 e and Additional file 2: Table S2 ). The expressions of Sept4 , Kcng4 , Unc13c and Prkcg genes in the TgW group, Edrg2 and Crh genes in the TgL group, Ccl2 , Nupr , H2-Q4 , Ccl12 , and Slc38a3 genes in the TgW vs TgL group were significantly down-regulated. Wh6 and LHG strains may manipulate host behaviors with different patterns of gene regulation Based on our PPI results, we found that compared to the Con group, the up-regulated key candidates Cxcl10 , Cxcl0 , Ccl5 , Ccl2 and Fcgr4 in the TgW group were related to the positive regulation of monocyte chemotaxis, and the H2-Eb1 , H2-Aa , H2-Ab1 and Cd74 were related to MHC class II-mediated exogenous antigen processing and presentation, and Tap1 was related to antigen processing and transport (Fig. 6 a). The down-regulated key candidates Unc13c , Prkcg , Sept4 , Kcng4 , etc ., were all related to the function of synaptic transmission (Fig. 6 b). Whereas in the TgL group, compared with the Con group, although the up-regulated key candidates H2-Aa , H2-Eb1 , H2-Ab1 , and Cd74 were also related to MHC class II-mediated exogenous antigen processing and presentation; the H2-Q7 , H2-D1 and B2m2 were related to MHC class I molecules. Besides, Ccl5 , Cxcl0 , and Irgm2 were related to the regulation of T cell chemotaxis (Fig. 6 c). Moreover, the down-regulated key candidates such as Crh and Ndrg2 , were related to neurodegenerative diseases (Fig. 6 d). Particularly, compared to the TgW group, the up-regulated key candidates Nr4a1 , Nr4a2 , Egr1 , and Egr2 in the TgL group were related to the cell response to corticotropin-releasing hormone stimulus, early growth response, N -terminal and NAB family, and nuclear glucocorticoid receptor binding. Simultaneously, Homer1 , Itpr1 , Prkcg , and Atp2b2 were related to postsynaptic cytosol and platelet calcium homeostasis, and Arc and Dlg4 were related to the regulation of synaptic plasticity (Fig. 6 e). Further, the down-regulated key candidates such as Ccl2 , Ccl12 , Ccl7 , were related to the regulation of natural killer cell chemotaxis, and Nupr1 , Slc38a3 , Ifi209 , Sfrp1 were related to the inhibition of SOCS3-mediated cell signaling and participate in the inhibition of inflammation (Fig. 6 f). Discussion T. gondii , a typical representative manipulator parasite [34, 35], can significantly affect the psychological behavior of the host and thus threaten public health [36, 37, 38]. It has been proved that neurogliocytes could be continuously activated by T. gondii infection, and the infected mice could present a marked decrease in microglia population compared to non-infected group, both with the ME49 clonal strain and TgCkBrRN2 atypical strain (CK2) [39]. Therefore, we have established in vitro infection models of Wh6 and LHG strains by using BV2 cell line, to better understand their invasion ability to microglia. Our current results demonstrated that Wh6 has a significantly more intensive ability than LHG in terms of microglia invasion and intracellular proliferation in vitro . Consistently, it has been proved that in the mice infected with TgCtWH6 strain, the hosts appeared apparently cognitive behavioral disorders, and the neuronal apoptosis, Aβ deposition, and neuroinflammatory response involved in microglia polarization may be the molecular and cellular mechanisms [40]. We have also found that the Wh6-infected mice showed significant cognitive impairment, speculating that the increased cognitive impairment caused by WH6 may be related to its stronger ability to microglia invasion. Moreover, our results also found that LHG had a stronger brain cyst formation ability in the infected mice, as well as an increased anxiety-like behavioral disorder during the chronic infection phase. Thus, our results further remind us that there may be a direct correlation between the brain cyst formation ability and the degree of behavioral abnormalities. Simultaneously, our ONT RNA-seq results revealed that the significantly up-regulated DETs in the two infected groups were primarily related to immune response and pathogen infection defense-related pathways, also consistent with the previous studies [41]. However, these studies mainly focused on the intensive virulent type I strain ( eg . RH) and the moderately virulent type II strains ( eg . Prugniaud and ME49) [42, 43, 44]. We believe our results would provide a necessary supplement for better understanding the mechanism of hosts' mental and behavioral disorders manipulated by T. gondii strains prevalent in China with different genotypes. Importantly, we also found that T. gondii strain with different genotypes could regulate host neuroinflammatory responses through different pathways. For instance, Wh6 and LHG may mediate the host neuroinflammatory response by up-regulating the differential MHC class molecules. Further, our results showed that genes related to synaptic transmission function were primarily down-regulated by Wh6 whereas genes related to neurodegenerative diseases were significantly down-regulated by LHG. In addition, we found that the mortality of LHG-infected mice had a significant gender preference in the acute infection stage. As a matter of fact, it has been indicated that the mortality of female mice infected with T. gondii was generally higher than that of male mice, and the reason may be the hypothesis that female mice could produce more inflammatory cytokines in response to T. gondii infection [45]. Our results precisely indicated that in the TgW vs TgL comparable group, the enrichment of response to IL-8 in the top 10 GO analysis in TgL group was more significant, indicating that the previous hypothesis is reasonable. As to the selected key candidates in the present study, the significant DETs in the different infected groups could also reflect the differences in the manipulation mechanism. For example, genes associated with positive regulation of monocyte chemotaxis, such as Fcgr4 , H2-Aa , H2-Ab1 , and Cd74 , were up-regulated in the TgW group, while genes associated with positive regulation of T cell chemotaxis, such as H2-Q7 and H2-D1 , were up-regulated in the TgL group. Since Fcgr4 , low-affinity immunoglobulin gamma Fc region receptor IV, has been reported to be up-regulated in leishmaniasis- infected mice, and ligation of FCGR4 by antigen-IgE(b) immune complexes could promote macrophage-mediated phagocytosis, antigen presentation to T cells, and proinflammatory cytokine production [46], so it has also been used to be a potential diagnostic marker for cutaneous leishmaniasis [47]. Secondly, the up-regulated H2-Q7 (i.e., H-2 class I histocompatibility antigen, Q7 alpha chain) belongs to the adaptive immune MHC I family response class, and it has been proved to participate in foreign antigen presentation, endogenous processing, and presentation of process polypeptide antigens, thus could promote the production of immune factors by T cells, and then enhance adaptive immunity [48]. These key candidates indicate that Wh6 strain was more likely to stimulate the phagocytosis and killing of the host central immune cells, whereas the LHG strain was more immunogenic to the host central immune system. This may be responsible for comprehending why LHG has a higher ability for brain cyst formation. Besides, there are several previous studies showed that WH6 strain could cause neuroinflammation, synaptic loss, and cognitive deficits in C57BL/6J mice, and the behavioral disorders phenotype were perfectly consistent with our study [49, 50] Simultaneously, in Tao' study, the tachyzoites of WH6 have been verified that they could indirectly induce APP protein production in HT22 by polarized BV2 cells, and the expression of CD80, pro-inflammatory factors, notch, and hes1 could be enhanced in the infected BV2 [49]. Moreover, the RNA-seq results in Wu' study [51] have also indicated that the WH6 infection could trigger extensive neuroinflammation in the prefrontal cortex, and the related genes such as Fcgr4 , Ccl22 and H2-D1 were highly consistent with our present research. As to the down-regulated key candidates, genes related to synaptic transmissions such as Unc13a , Prkcg , Sept4 , and Kcng4 have been selected from the TgW group, while genes related to neurodegeneration such as Crh and Ndrg2 were selected from the TgL group. Firstly, it is well known that the Unc13 (Protein unc-13 homolog C) family is critical in regulating neurotransmitter release and synaptic plasticity in CNS. Unc13a is involved in several related processes including glutamatergic synaptic transmission, synaptic vesicle maturation, and the regulation of synaptic transmission at parallel fiber-Purkinje cell synapses [52]. Secondly, Ndrg2 is an N -myc downstream regulatory gene family member. It is primarily expressed in the astrocytes and involved in the pathogenesis of many neurological diseases, such as stroke, neurodegenerative diseases (Alzheimer's and Parkinson's diseases), and psychiatric disorders (depression and attention deficit hyperactivity disorder) [53]. This would be auxiliary to explain why the infected animals showed a loss of natural fear of predators, anxiety behaviors, and memory impairments. Importantly, expression of genes related to synaptic plasticity and neurological diseases such as Arc , Homer1 , and Itpr1 in the TgL group were significantly up-regulated compared to the TgW group. Arc is an activity-regulated cytoskeleton-associated protein. It can regulate synaptic strength through multiple mechanisms and is essential for memory consolidation and learning [54]. In the meanwhile, the expression of natural killer cell chemotaxis-related genes such as Ccl2 , Ccl12 , H2-Q4, and Slc38a3 in the TgL group was lower expressed than that of the TgW group. Particularly, CCL2, a ligand for C-C chemokine receptor CCR2, could act on dopaminergic neurons to increase their excitability, dopamine release, and motor activity, as well as increase NMDA-mediated synaptic transmission in neurons containing dopamine D1 and D2 receptors [55]. As a result, our founding may provide a further understanding of why LHG could induce more severe anxiety-like behaviors. Mental and behavioral manipulation mechanisms of difference T. gondii strains to their host are complex. Although our results promote a hypothesis that difference of neuroinflammatory response and synaptic transmission may be the key pathways for the difference of invasion and mental manipulation ability between the two T. gondii strains. However, the accurate molecular mechanisms need to be further verified. Conclusion In conclusion, both Chinese I Wh6 strain and Chinese III LHG strain could cause cognitive and spatial memory impairment, and anxiety-like behaviors in the hosts, but the anxiety caused by LHG appeared more obvious. Immune response and synaptic transmission were significantly enriched and may be the major influenced pathways. Besides, our results indicated that genes related to synaptic transmission such as Unc13c, Prkcg, Sept4, and Kcng4 may be the main factors responsible for Wh6 mental and behavioral manipulation; while genes related to neurodegeneration such as Ndrg2 and Arc , as well as Ccl2 , maybe the key factors for explaining the stronger mental manipulation ability of LHG strain (Graphical abstract). Abbreviations T. gondii : Toxoplasma gondii ; Wh6: Chinese cat-derived type I TgCtWh6 strain of T. gondii ; LHG: human type III TgCtLHG strain of T. gondii ; DETs: differentially expressed transcripts; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: protein–protein interaction; qPCR: quantitative polymerase chain reaction; AIDS: Acquired Immunodeficiency Syndrome; CNS: Central Nervous System; DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum; Con: control group; TgW: Wh6-infected; TgL: LHG-infected; OFT: open-field: test; YM: Y-maze; PBS: sterile phosphate-buffered saline; SEMs: standard errors of the means; ONT: Oxford Nanopore Technologies; BPs: biological processes; EB: Epstein–Barr; GABA: Gamma-aminobutyric acid; HIV: Human Immunodeficiency Virus; AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid; cGMP-PKG: cyclic guanosine monophosphate-protein kinase G; MHC: major histocompatibility complex; TAP1: Transporter associated with antigen processing 1. Declarations Supplementary information Supplementary data to this article can be found online at: xxxDeclarations Ethics approval and consent to participate All animal experiments were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals (issued by the Council for the International Organizations of Medical Sciences), as well as the guidelines set by the Institutional Animal Care and Use Committee of Shandong First Medical University (approval number: w202103030088) and by Animal Research: Reporting of in vivo Experiments (ARRIVE). Author ’s Contributions KY, GHZ and JPC conceived the study, designed the experiments and critically revised the manuscript. BBZ, HJD, HS and XXM performed the experiments, analyzed the data and drafted the manuscript. HHX, WJZ, YNL and CX participated in the implementation of the study. Funding This work was supported by the Parasitic Pathogens of National Health Committee and Vector Biology Key Laboratory Open Research Topic [grant number NHCKFKT2022-15]; the Natural Science Foundation of Shandong Province [grant number ZR2022MH197]; the Medical and Health Science and Technology Program of Shandong Province [grant number 202401050156]; and the Taishan Scholars Project of Shandong Province [grant number tsqn202103186]. Data Availability Statement The data presented in this study are included in the article and its Supplementary Material. Further any further inquiries, please contact the corresponding author. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Acknowledgments The authors gratefully acknowledge Prof. Jilong Shen (Anhui Medical University, Hefei, China) for generously providing the Chinese I T. gondii strain- TgCtwh6 and the Chinese III T. gondii strain- TgCtLHG . 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Arrows indicate typical \u003cem\u003eT. gondii\u003c/em\u003e tachyzoites invading cells during the co-culturing period. \u003cstrong\u003e(b) \u003c/strong\u003eInfected mice and cysts formed in mouse brain tissues (magnifications: 100×; scale bar: 50 μm). Arrows indicate typical cysts in the brain tissue. \u003cstrong\u003e(c) \u003c/strong\u003eComparison of the number of cysts between Wh6 and LHG infected brain tissues. Values are presented as means ± SEMs. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/58d969eb4a5a2d071f1e2570.png"},{"id":81063653,"identity":"60217fa0-154f-4851-8866-531025ccc48c","added_by":"auto","created_at":"2025-04-21 20:11:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":606063,"visible":true,"origin":"","legend":"\u003cp\u003eThe behavior analysis results of mice infected with or without WH6 and LHG strain of \u003cem\u003eT. gondii\u003c/em\u003e. Total distance traveled \u003cstrong\u003e(a)\u003c/strong\u003e, (\u003cem\u003eF\u003c/em\u003e = 0.274, \u003cem\u003eP\u003c/em\u003e = 0.764, LSD test) Autonomous alteration rate \u003cstrong\u003e(b)\u003c/strong\u003e, (\u003cem\u003e F\u003c/em\u003e= 11.757, \u003cem\u003eP\u003c/em\u003e = 0.001, LSD test)and Motion trajectory diagram \u003cstrong\u003e(c)\u003c/strong\u003e. Results of OFT without provocation. total distance \u003cstrong\u003e(d)\u003c/strong\u003e, Average velocity \u003cstrong\u003e(e)\u003c/strong\u003e, Time of immobility \u003cstrong\u003e(f)\u003c/strong\u003e, Frequency of entries into the central zone (\u003cstrong\u003eg)\u003c/strong\u003e, Distance moved in the central zone (\u003cstrong\u003eh)\u003c/strong\u003e, Time spent in the central zone \u003cstrong\u003e(i)\u003c/strong\u003e, and Motion trajectory diagram \u003cstrong\u003e(j)\u003c/strong\u003e. Investigation time of OFT with cat urine.\u003cstrong\u003e \u003c/strong\u003eInvestigation time \u003cstrong\u003e(k)\u003c/strong\u003e, and Motion trajectory diagram\u003cstrong\u003e (l)\u003c/strong\u003e. Data are presented as means ± SEM. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/ab6adde327c02b3f0fd30adb.png"},{"id":81064308,"identity":"763fdeaf-f801-4b65-92bd-6d214fff149a","added_by":"auto","created_at":"2025-04-21 20:35:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":279643,"visible":true,"origin":"","legend":"\u003cp\u003eMA plots of DETs in Con vs. TgW (\u003cstrong\u003ea)\u003c/strong\u003e, Con vs. TgL (\u003cstrong\u003eb)\u003c/strong\u003e, and TgW vs. TgL (\u003cstrong\u003ec)\u003c/strong\u003e groups.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/e5c01d8e7f6ef18a0e88ee2e.png"},{"id":81063658,"identity":"7eeff95b-8f37-49ac-a5c3-f2298c69cb58","added_by":"auto","created_at":"2025-04-21 20:11:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":633593,"visible":true,"origin":"","legend":"\u003cp\u003eTop 10 up-regulated and down-regulated DETs as shown by GO and KEGG enrichment analysis in\u003cstrong\u003e \u003c/strong\u003eCon vs. TgW \u003cstrong\u003e(a),\u003c/strong\u003e Con vs. TgL \u003cstrong\u003e(b),\u003c/strong\u003eand TgW vs. TgL \u003cstrong\u003e(c) \u003c/strong\u003egroups.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/cc37fcf5c425265fe2e08a37.png"},{"id":81063655,"identity":"f67296dc-b2d8-4517-a5c2-08b137d9c1ca","added_by":"auto","created_at":"2025-04-21 20:11:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":313839,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmaps of genes corresponding to key DETs and qPCR validation results of key candidates genes. Gene expression profiles of up-regulated and down-regulated DETs in\u003cstrong\u003e \u003c/strong\u003eCon vs. TgW \u003cstrong\u003e(a)\u003c/strong\u003e, Con vs. TgL \u003cstrong\u003e(b)\u003c/strong\u003e, and TgW vs. TgL \u003cstrong\u003e(c) \u003c/strong\u003egroups. qPCR results of the up-regulated key candidate DETs\u003cstrong\u003e (d)\u003c/strong\u003e and\u003cstrong\u003e \u003c/strong\u003edown-regulated \u003cstrong\u003e(e)\u003c/strong\u003e. Data are presented as means ± SEMs. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/b3c2c88aedd49eae8dc01e3c.png"},{"id":81063896,"identity":"3a1272d1-e50f-4837-9496-34353562bcc3","added_by":"auto","created_at":"2025-04-21 20:19:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":491677,"visible":true,"origin":"","legend":"\u003cp\u003ePPI network of DETs-encoded proteins. Up-regulated DETs in\u003cstrong\u003e \u003c/strong\u003eCon vs. TgW \u003cstrong\u003e(a)\u003c/strong\u003e, Con vs. TgL \u003cstrong\u003e(c)\u003c/strong\u003e, and TgW vs. TgL \u003cstrong\u003e(e)\u003c/strong\u003e groups. Down-regulated DETs in Con vs. TgW \u003cstrong\u003e(b)\u003c/strong\u003e, Con vs. TgL \u003cstrong\u003e(d)\u003c/strong\u003e, and TgW vs. TgL \u003cstrong\u003e(f) \u003c/strong\u003egroups. * represents genes of key candidate DETs validated by qPCR; # denotes genes with no changes in key candidate DETs validated by qPCR.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/2057eec91ae8f2d33ffce71a.png"},{"id":83783472,"identity":"d712ae29-7b66-4419-b698-77f14fe4fea2","added_by":"auto","created_at":"2025-06-02 16:11:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4230540,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/97c07baa-5176-434e-b52c-563cefcdb1d6.pdf"},{"id":81063897,"identity":"b655dd40-d4a8-408d-9d01-b4aaedaefa9c","added_by":"auto","created_at":"2025-04-21 20:19:22","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5827252,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/6940e24a6b9e4b091e8c65aa.tif"},{"id":81064165,"identity":"6cdde64f-719b-48ae-a14e-191fc231882d","added_by":"auto","created_at":"2025-04-21 20:27:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22067,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/67c9681ff8e91b09177af94a.docx"},{"id":81063895,"identity":"f5cd834f-f0af-4768-9621-d5ee5f53a5f4","added_by":"auto","created_at":"2025-04-21 20:19:21","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21832,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6075517/v1/384ba9ca7e8ea9c6e15e2075.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of latent infection of Toxoplasma gondii strains with different genotypes on mouse behavior and brain transcripts","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eT. gondii\u003c/em\u003e is an intracellular apicomplexan parasite infecting nearly all warm-blooded animals worldwide [1, 2]. About 30\u0026ndash;50% of the global population has been exposed to or infected with \u003cem\u003eT. gondii\u003c/em\u003e [3]. \u003cem\u003eT. gondii\u003c/em\u003e is highly prevalent in Western Europe, South America, and Africa [4, 5], with infection rates ranging from 10% in the United States to \u0026gt;\u0026thinsp;50% in France, Colombia, and Brazil [6, 7, 8]. Moreover, a 2018 survey on toxoplasmosis in China demonstrated that the \u003cem\u003eT. gondii\u003c/em\u003e infection rate among the Chinese population was 8.2%, and 23.7% among livestock [9]. Because of its high prevalence and widespread distribution in its intermediate hosts including livestock, poultry, and pets, \u003cem\u003eT. gondii\u003c/em\u003e is increasingly recognized as a considerable risk to human health and the livestock industry.\u003c/p\u003e \u003cp\u003eTachyzoites could rapidly invade host's blood and various organs including eyes and internal organs, resulting in serious acute infections, particularly in immunodeficient patients. For instance, toxoplasmosis-induced encephalitis is a common cause of death in patients with Acquired Immunodeficiency Syndrome (AIDS) [10]. Although the clinical symptoms in infected individuals with normal immune function are not apparent in the acute phase, tachyzoites could also transform into bradyzoites and tissue cysts in the latent phase. Notably, cerebral cysts, the most common \u003cem\u003eT. gondii\u003c/em\u003e-associated tissue cyst type, could persistently infect brain tissues, leading to mental and behavioral disorders [11, 12]. In rodent infection models, it can trigger various behavioral disorders including loss of natural fear, an increased desire to explore, and a decline in cognitive function [13, 14, 15]. Further, it has been proved that neuroinflammation and synaptic alterations were associated with the behavioral abnormal post-chronic infections with \u003cem\u003eT. gondii\u003c/em\u003e strains, for example, the synaptic proteins EAAT2 and GABAAα1, which are involved in excitation/inhibition balance in Central Nervous System (CNS), could be down-regulated after infection, and accompanied with increased microglia activation [16, 17]. In some cases, different phenotypes such as depression, anxiety, and reduced motor capacity have also been reported [18, 19, 20]. So far, there are already more than 300 publications have shown that various types of psychiatric disorders, ranging from schizophrenia to depression, suicidal tendencies, Alzheimer's disease, road rage-induced traffic accidents, and even positive behaviors (e.g., entrepreneurship and risk-taking) are significantly associated with \u003cem\u003eT. gondii\u003c/em\u003e infection [21, 22]. However, the mental behavioral manipulation patterns caused by different \u003cem\u003eT. gondii\u003c/em\u003e strains with different genotypes, as well as the different pathogenic characteristics, particularly for Chinese \u003cem\u003eT. gondii\u003c/em\u003e isolates, still remain unclear [13, 14, 23].\u003c/p\u003e \u003cp\u003eUp to now, there are 12 genotypes of Chinese \u003cem\u003eT. gondii\u003c/em\u003e strains have been identified, and the dominant strain \u003cem\u003eChinese I\u003c/em\u003e (\u003cem\u003eToxoDB#9\u003c/em\u003e) occupied 66.36% of all genotypes, followed by type I and II (variants) [24, 25, 26]. In particular, the \u003cem\u003eChinese III\u003c/em\u003e strain, also widespread in humans and domestic animals, is phylogenetically close to the typical type I virulent strains such as RH and GT1 [27] Although its virulence is weaker, it is more likely to form brain cysts than \u003cem\u003eChinese I\u003c/em\u003e isolates [28, 29]. However, there are neither study has reported the phenotype of behaviors of hosts infected with \u003cem\u003eChinese III\u003c/em\u003e strains, nor has compared the manipulation patterns and mechanisms of different Chinese \u003cem\u003eT. gondii\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eTherefore, in the present study, we have established both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e infection models with \u003cem\u003eTgCtwh6\u003c/em\u003e (Wh6; feline-sourced Ⅰ\u0026times;Ⅱ hybrid strain) and \u003cem\u003eTgCtLHG\u003c/em\u003e (LHG; human-sourced type III strain), respectively. Based on these, we have analyzed the differences in host symptoms in both acute and latent infection phases and assessed the behavioral differences as well as the full-length cerebral cortex transcript expression before and after chronic infection. Our findings may provide a basis for an in-depth interpretation of parasite-mediated mental and behavioral manipulation mechanisms with different genotypes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and parasites\u003c/h2\u003e \u003cp\u003e \u003cem\u003eTgCtWh6\u003c/em\u003e (Wh6, a Chinese Ⅰ\u0026times;Ⅱ hybrid genotype strain, isolated from a stray cat in Wuhan, China) and \u003cem\u003eTgCtLHG\u003c/em\u003e (LHG, a type III genotype strain, isolated from the peripheral blood of a patient with toxoplasmosis) were kindly provided by Prof. Jilong Shen at Key Laboratory of Microbiology and Parasitology, Anhui Medical University (Hefei, Anhui, China). Brain cysts of Wh6 and LHG were continuously transmitted to the animal hosts BALB/c mice via cyst gavage [30].\u003c/p\u003e \u003cp\u003eSpecific pathogen-free C57BL/6J mice, aged 6\u0026ndash;8 weeks, were purchased from Pengyue Experimental Animal Breeding (Jinan, Shandong, China; animal license number: SCXK(Lu) 20220006). All animals were maintained under a constant temperature (22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (55% \u0026plusmn; 5%) under a 12 h day-night cycle, with free access to food and water. The drinking water was sterilized through high-pressure disinfection, and the feed and bedding were sterilized using ultraviolet light.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBV2 cells were routinely cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, USA) at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. When the passage density was reached, all cells were digested with 0.25% EDTA-trypsin, divided into flasks at a 1:2 ratio, and cultured at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e until 70\u0026ndash;80% of the cells fused. Next, the medium was replaced with fresh high-glucose DMEM containing 3% fetal bovine serum. After 12 h, the cells were used in an \u003cem\u003ein vitro\u003c/em\u003e experiment with \u003cem\u003eT. gondii\u003c/em\u003e tachyzoites. In total, six-cell culture dishes (35 mm \u0026times; 10 mm) were placed in sterile conditions and numbered, and 2.4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were inoculated into each dish. After 12 h of culture at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e, the medium was replaced, and the cells were allowed to grow to 85% confluency. Tachyzoites were co-cultured with BV2 cells at a 1:2 ratio, and the infected cells were observed for invasion status at 12, 24, 48, and 72 h after infection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction of chronic infection mouse model\u003c/h3\u003e\n\u003cp\u003eIn total, 60 female and 60 male C57BL/6J mice, aged 6\u0026ndash;8 weeks, were used to establish chronic infection models. Animals were divided into three groups: control group (Con), Wh6-infected group (TgW), and LHG-infected group (TgL). Each group included 20 female and 20 male mice. Each infection group was gavaged with 0.2 mL of cerebral tissue suspension containing 10 cysts, and the control group was gavaged with 0.2 mL of saline. After 45 days of inoculation, one mouse from each group was randomly selected and euthanized to obtain brain tissue. Besides, cysts were observed under a microscope to determine infection effectiveness.\u003c/p\u003e\n\u003ch3\u003eClinical observation\u003c/h3\u003e\n\u003cp\u003eWe observed clinical symptoms exhibited by tachyzoite-infected mice daily over 0\u0026ndash;42 days after infection. For each infected mouse, the onset and duration of acute symptoms, such as back arching, erect hair, tremors, limb weakness, and eye disturbances, were recorded. The sex, survival time, and number of dead mice were also documented.\u003c/p\u003e\n\u003ch3\u003eBehavioral testing\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eY-maze test (YM)\u003c/h2\u003e \u003cp\u003eEach mouse was placed in the starting arm of a Y-maze and allowed to explore the three arms. The exploration behavior of a test mouse was recorded by a camera; in particular, we recorded the total distance traveled, the number of entries into each arm, and spontaneous alternation [= (total number of alternations/number of maximum alternations) \u0026times; 100%].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOpen-field test (OFT)\u003c/h2\u003e \u003cp\u003eAn OFT was conducted using a camera to detect the movements of animals in a 50 \u0026times; 50 cm\u003csup\u003e2\u003c/sup\u003e arena in a quiet, soundproof, dimly lit (\u0026asymp;\u0026thinsp;20 lx) room. A mouse was placed in the center of the arena and allowed to move freely for 10 min. The movements of each mouse in each of the three groups were recorded using an automatic video tracking system for 1 min. In particular, we recorded the total distance traveled, average speed, immobility time, number of entries into the central area, distance traveled within the central area, and time spent in the central area.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePredator Odor-Induced OFT\u003c/h3\u003e\n\u003cp\u003eThis test was used to evaluate the host's fear response to predators. The method was similar to the OFT, but the urine of cat was placed sequentially in one corner of each compartment to assess how much time the mice spent in the urine-exposed areas.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBrain tissue collection and\u003c/b\u003e \u003cb\u003eT. gondii\u003c/b\u003e \u003cb\u003ecysts counting\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter 45 days of infection, the mice were anesthetized, euthanized, and immediately dissected to remove the whole brain. The brain tissue was washed with sterile phosphate-buffered saline (PBS) and placed in a Keeper preservation solution.\u003c/p\u003e \u003cp\u003eA random sample of mice from the infected groups was euthanized under sterile conditions with ether anesthesia, and the whole brain tissue was segmented along the longitudinal axis into left and right hemispheres, with the cerebral cortex, hippocampus, and olfactory bulb separated. Each part was thoroughly homogenized in 2 mL of PBS. Next, 10 \u0026micro;L of the homogenate was used to prepare a smear. The number of \u003cem\u003eT. gondii\u003c/em\u003e cysts was counted under a microscope (400\u0026times; magnification), and for each mouse, an average value was calculated from three smears.\u003c/p\u003e\n\u003ch3\u003eRNA extraction\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA extraction\u003c/div\u003e \u003cp\u003eAfter completion of all behavior tests, the whole brains of all tested mice were excised and immediately frozen in liquid nitrogen. Since cysts are predominantly localized in cortical areas of the brain [31], the whole cortical regions of a tested mouse were separated and used to be an RNA sample. The total RNA in each sample was extracted using TRIzol reagent (Thermo Fisher, USA), according to the manufacture's instructions. The total absorbance (A\u0026thinsp;=\u0026thinsp;A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e230\u003c/sub\u003e) was detected to analyze the purity of RNA, and the Aglient 2100 Bioanalyzer was used to detect RNA concentration and integrity.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLibrary construction and Nanopore RNA sequencing\u003c/h2\u003e \u003cp\u003eWe used Oxford Nanopore Technologies-based single-molecule real-time sequencing technology (i.e., ONT RNA-seq) for sequencing full-length RNA transcripts. Total RNA was extracted from mouse brain tissue, and mRNA was obtained through magnetic bead enrichment. The library was subsequently constructed, and the high-throughput sequencing was performed by Biomarker Technologies Co., LTD. The raw sequence was evaluated for quality and filtered to remove adapter sequences, short segments, and low-quality sequences; finally, high-quality read lengths were analyzed, and data on differential representative transcripts were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFunctional enrichment analysis\u003c/h2\u003e \u003cp\u003eDifferentially expressed transcriptome data were obtained through quantitative analysis of transcriptome data from the brains of infected and control mice. The resulting sequences of differently expressed transcripts were compared with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes libraries (KEGG) to obtain relevant functional annotations and differentially expressed transcripts (DETs). To understand the changes in transcription levels in infected mice, we analyzed DETs with the following conditions: fold change (i.e., fold differences in DETs expression) is \u0026ge;\u0026thinsp;1.5, and \u003cem\u003eP\u003c/em\u003e (i.e., significance screening index for DETs) is \u0026lt;\u0026thinsp;0.05. The classification and enrichment of DETs were performed to GO functional annotation and KEGG pathway analyses in order to select the top 10 up-regulated and top 10 down-regulated DETs. The \u003cem\u003eP\u003c/em\u003e values for these analyses were calculated using edge R, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered to indicate statistically significant enrichment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein-protein interaction and cluster analysis\u003c/h2\u003e \u003cp\u003eThe potential interactions between the encoded proteins were retrieved from the string database, the protein-protein interaction (PPI) networks were constructed, and the clustering analysis of protein populations was performed using clustering options [32].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistics analysis\u003c/h2\u003e \u003cp\u003eAll data, expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (SEMs), were analyzed using GraphPad Prism (version 8.0). We used the Shapiro-Wilk normality test followed by Student's \u003cem\u003et-test\u003c/em\u003e to compare the data from the two groups. Moreover, One-way ANOVA analysis followed by the post hoc Turkey test was used to analyze the effects of \u003cem\u003eT. gondii\u003c/em\u003e infection. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eWh6 tachyzoites are more invasive and proliferative than LHG tachyzoites within glial cells\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBy the continuous detection under the inverted microscope, most tachyzoites of either strain could adhere to BV2 cell surfaces at 12 h of co-culturing, with only a few of them entering the cytoplasm. Almost all of the tachyzoites could complete cell invasion at 24 h after infection. At 48 h of co-culturing, the parasites began to divide, some Wh6-infected BV2 cells demonstrated a protrusion filled with the parasites on their membranes. By 72 h after infection, many parasites were released and began to start a new round of invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, upward arrow). However, the tachyzoites of LHG could not lead to cell membrane protrusion or parasite release until 72 h after co-culturing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, downward arrow). Thus, our results showed that Wh6 has stronger invasive and proliferative ability in BV2 cells than LHG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe infection symptoms caused by Wh6 and LHG tachyzoites, and the number of brain cysts were different\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAt 5 days after infection, the infected mice began to exhibit acute symptoms such as hunched back, piloerection, tremor, limb weakness, eye discoloration, and decreased activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This acute onset phase cloud lasts for 20 days after infection. In general, TgW mice tended to have more severe eye damage, while TgL mice were more likely to demonstrate hind limb weakness (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We found that all infected mice could demonstrate cyst formation in brain tissues at 21 days after infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), but the number of brain cysts in the TgL group was significantly more than those in the TgW group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Surprisingly, we also found that all of the dead mice were female, and most of them belonged to the TgL group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As a result, we speculated that LHG strain may have a sex bias for host fatality.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eContinuous symptom observations of mice in TgW and TgL groups (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMouse sex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eT. gondii\u003c/em\u003e strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSurvival\u003c/p\u003e \u003cp\u003e(days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDeath rate\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c9\" namest=\"c5\"\u003e \u003cp\u003eAcute symptom rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHogback\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePiloerection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eShiver\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHind leg weakening\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWhitening of eyes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWh6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50 (11/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12.5 (2/20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLHG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50 (12/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWh6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.0 (2/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87.5 (18/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (1/20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLHG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.0 (13/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csub\u003ea\u003c/sub\u003e Death rate = (number of deaths / number of infected individuals \u0026times; 100%).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferent genotypes of\u003c/b\u003e \u003cb\u003eT. gondii\u003c/b\u003e \u003cb\u003estrain could cause different types of mental and behavioral disorders\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eThe Y-maze results showed that chronic T. gondii infection impaired the spatial working memory ability of mice.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAlthough the total distance traveled in the YM test did not differ significantly among the TgW, TgL, and Con groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), the autonomous alternation rate decreased significantly in both infected groups compared to the Con group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), indicating that both WH6 and LHG could impair the spatial working and short-term memory ability of the hosts in the chronic infection phase, with a similar degree. A representative motion trajectory diagram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eThe OFT results showed that the TgL group had more severe motor impairments and higher anxiety levels.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCompared with the Con group, both TgW and TgL groups exhibited significantly lower average speeds and longer immobility times, however, the TgW group showed significantly longer total distance traveled (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f), and an increase in the total number of entries into the central zone but a reduction in the total distance traveled and time spent in the central zone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-i). Thus, the chronic infection may cause slight motor impairments along with anxiety-like behavior in the hosts. Compared with TgW, the TgL group demonstrated a slightly shorter total distance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), lower average speed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly higher immobility time (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u0026ndash;f), indicating that LHG strain had relatively more severe motor impairments, consistent with our results in the acute infection phase. In particular, mice in the TgL group showed fewer central zone entries but slightly longer time spent in the central zone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-i), suggesting that LHG strain may be more likely to cause hosts' anxiety. A representative motion trajectory diagram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej.\u003c/p\u003e \u003cp\u003e \u003cem\u003eThe cat urine OFT results showed that both TgW and TgL mice had the loss of natural fear of predators, while TgW group was more significant.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe collected urine samples from domestic cats and conducted a predator-induced mineshaft OFT. The results showed that all the infected mice spent significantly longer time in the urine-containing area than the control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek), consistent with the previous results [31, 33]. However, mice in the TgW group appeared more significant fear loss degree (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) than those of the TgL group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A representative motion trajectory diagram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of DETs among TgW, TgL, and Con groups\u003c/h2\u003e \u003cp\u003eThe third-generation high-throughput ONT RNA-seq results demonstrated that the TgW and TgL groups included 1,789 and 1,119 DETs compared with the Con group, respectively. The TgW and TgL groups included 1,659 and 1,089 up-regulated DETs, respectively; and 130 and 30 down-regulated DETs, respectively. In the comparison of the TgW and TgL groups, 466 DETs were detected, of which 223 were up-regulated and 243 were down-regulated, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChronic infection can intensively influence the immune response and synaptic transmission-related processes of the host\u003c/h2\u003e \u003cp\u003eCompared to the Con group, the top 10 up-regulated BPs in the TgW group were immune response, response to protists, cellular response to interferon β, antigen processing and presentation, inflammatory response, cellular response to interferon γ, innate immune response, defense response, defense response to viruses, and response to bacteria. The top 10 up-regulated KEGG pathways were Epstein\u0026ndash;Barr (EB) virus infection, allogenic transplant rejection, antigen processing and presentation, phagosome, xenograft host defense, autoimmune thyroid disease, viral myocarditis, type I diabetes, leishmaniasis, and cell adhesion molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, left\u003cb\u003e)\u003c/b\u003e. The top 10 down-regulated BPs were the Gamma-aminobutyric acid (GABA) signaling pathway, protein homo-oligomerization, morphogenesis of the camera-shaped eye development, GABAergic synaptic transmission, 9-cis retinoic acid biosynthesis process, chloride ion transport, vesicle fusion-positive regulation, inhibitory synapse assembly, hair cycle regulation, and calcium-dependent exocytosis positive regulation. The top 10 down-regulated KEGG pathways were nicotine addiction, retrograde endogenous cannabinoid signaling, morphine addiction, GABAergic synaptic transmission, neuroactive ligand-receptor interactions, taste transduction, dopaminergic synapses, synaptic vesicle cycles, epidermal growth factor receptor tyrosine kinase inhibitor resistance, and other types of o-glycan biosynthesis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, right). Taken together, these results demonstrated that infection with Wh6 mainly leads to up-regulation of processes involved in immune responses associated with pathogenic infection and down-regulation of processes related to synaptic signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimultaneously, compared to the Con group, the top 10 up-regulated BPs in the TgL group were immune response, cellular response to interferon β, cellular response to interferon γ, antigen processing and presentation, response to protists, defense response, response to bacteria, innate immune response, defense response to gram-positive bacteria, and inflammatory response. The top 10 up-regulated KEGG pathways were EB virus infection, allogenic transplant rejection, phagosome, antigen processing and presentation, xenograft host defense, viral myocarditis, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infection, type I diabetes, and leishmaniasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, left). The top 10 down-regulated BPs were the regulation of platelet-derived growth factor generation, regulation of vascular endothelial growth factor generation, negative regulation of the extracellular signal-regulated kinase 1 and 2 cascades, negative regulation of cell growth, membrane invagination, synaptic vesicle cycle, vesicle-mediated transport in synapses, positive regulation of adipocyte differentiation, negative regulation of hepatocyte differentiation, and negative regulation of forebrain neuron differentiation. The top 10 down-regulated KEGG pathways were alcohol addiction, chemokine signaling pathways, human cytomegalovirus infection, HIV type I infection, GABAergic synapses, circadian rhythms, opioid addiction, serotonergic synapses, cholinergic synapses, and glutamatergic synapses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, right). Similarly, these results indicated that infection with LHG would also lead to the up-regulation of immune responses, and the down-regulation of synaptic signaling, as well as processes related to cell differentiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLHG strain may cause enhanced variation of synaptic transmission and metabolic secretion\u003c/h2\u003e \u003cp\u003eCompared to the TgW group, the top 10 up-regulated BPs in the TgL group were the regulation of neuronal synaptic plasticity, response to nicotine, positive regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor activity, behavior, intracellular transport, cellular response to interleukin 8, positive regulation of synaptic transmission, regulation of calcium ion transport, long-term memory, and regulation of cation channel activity. The top 10 up-regulated KEGG pathways were aldosterone synthesis and secretion, glutamatergic synapses, calcium signaling pathways, pancreatic secretion, salivary secretion, gastric secretion, cosecretion and action of parathyroid hormones, retrograde endocannabinoid signaling, long-term potentiation, and cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, left). The top 10 down-regulated BPs were the cellular response to gamma interferon, antigen processing, and presentation, positive regulation of type II hypersensitivity reactions, inflammatory responses, acetylcholine receptor signaling pathways, immune responses, lipoprotein lipid oxidation negative regulation, cellular response to interferon β, fibroblast proliferation negative regulation, and complement activation. The top 10 down-regulated KEGG pathways were phagosomes, plant-pathogen interactions, allograft rejection, type 1 diabetes, autoimmune thyroid disease, \u003cem\u003eS. aureus\u003c/em\u003e infection, EB virus infection\u0026ndash;viral myocarditis, and systemic lupus erythematosus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, right). These results demonstrated that the regulation of hosts' synaptic transmission and metabolic secretory function may be enhanced by LHG strain, yet the inflammatory immune responses showed more weakness in the TgL group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eVerification of the key DET candidates\u003c/h2\u003e \u003cp\u003eDETs with high expression changes, top GO annotation, and top KEGG enrichment were selected to obtain heatmaps of gene expression profiles corresponding to the top 10 key candidate DETs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). Among them, the first four up-regulated key candidates were selected for qPCR verification in each group, and the results showed that most of the gene expression was consistent with our ONT RNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In particular, the mRNA expression of \u003cem\u003eH2-Aa\u003c/em\u003e and \u003cem\u003eFcgr4\u003c/em\u003e in the TgL group, \u003cem\u003eH2-Q7\u003c/em\u003e in the TgL group, and \u003cem\u003eArc\u003c/em\u003e and \u003cem\u003eItpr1\u003c/em\u003e in the TgW vs TgL group were significantly up-regulated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, the verification of down-regulated key candidates showed that a majority of the selected gene expression results were consistent with the DETs results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The expressions of \u003cem\u003eSept4\u003c/em\u003e, \u003cem\u003eKcng4\u003c/em\u003e, \u003cem\u003eUnc13c\u003c/em\u003e and \u003cem\u003ePrkcg\u003c/em\u003e genes in the TgW group, \u003cem\u003eEdrg2\u003c/em\u003e and \u003cem\u003eCrh\u003c/em\u003e genes in the TgL group, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eNupr\u003c/em\u003e, \u003cem\u003eH2-Q4\u003c/em\u003e, \u003cem\u003eCcl12\u003c/em\u003e, and \u003cem\u003eSlc38a3\u003c/em\u003e genes in the TgW vs TgL group were significantly down-regulated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eWh6 and LHG strains may manipulate host behaviors with different patterns of gene regulation\u003c/h2\u003e \u003cp\u003eBased on our PPI results, we found that compared to the Con group, the up-regulated key candidates \u003cem\u003eCxcl10\u003c/em\u003e, \u003cem\u003eCxcl0\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e and \u003cem\u003eFcgr4\u003c/em\u003e in the TgW group were related to the positive regulation of monocyte chemotaxis, and the \u003cem\u003eH2-Eb1\u003c/em\u003e, \u003cem\u003eH2-Aa\u003c/em\u003e, \u003cem\u003eH2-Ab1\u003c/em\u003e and \u003cem\u003eCd74\u003c/em\u003e were related to MHC class II-mediated exogenous antigen processing and presentation, and \u003cem\u003eTap1\u003c/em\u003e was related to antigen processing and transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The down-regulated key candidates \u003cem\u003eUnc13c\u003c/em\u003e, \u003cem\u003ePrkcg\u003c/em\u003e, \u003cem\u003eSept4\u003c/em\u003e, \u003cem\u003eKcng4\u003c/em\u003e, \u003cem\u003eetc\u003c/em\u003e., were all related to the function of synaptic transmission (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhereas in the TgL group, compared with the Con group, although the up-regulated key candidates \u003cem\u003eH2-Aa\u003c/em\u003e, \u003cem\u003eH2-Eb1\u003c/em\u003e, \u003cem\u003eH2-Ab1\u003c/em\u003e, and \u003cem\u003eCd74\u003c/em\u003e were also related to MHC class II-mediated exogenous antigen processing and presentation; the \u003cem\u003eH2-Q7\u003c/em\u003e, \u003cem\u003eH2-D1\u003c/em\u003e and \u003cem\u003eB2m2\u003c/em\u003e were related to MHC class I molecules. Besides, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCxcl0\u003c/em\u003e, and \u003cem\u003eIrgm2\u003c/em\u003e were related to the regulation of T cell chemotaxis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Moreover, the down-regulated key candidates such as \u003cem\u003eCrh\u003c/em\u003e and \u003cem\u003eNdrg2\u003c/em\u003e, were related to neurodegenerative diseases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eParticularly, compared to the TgW group, the up-regulated key candidates \u003cem\u003eNr4a1\u003c/em\u003e, \u003cem\u003eNr4a2\u003c/em\u003e, \u003cem\u003eEgr1\u003c/em\u003e, and \u003cem\u003eEgr2\u003c/em\u003e in the TgL group were related to the cell response to corticotropin-releasing hormone stimulus, early growth response, \u003cem\u003eN\u003c/em\u003e-terminal and NAB family, and nuclear glucocorticoid receptor binding. Simultaneously, \u003cem\u003eHomer1\u003c/em\u003e, \u003cem\u003eItpr1\u003c/em\u003e, \u003cem\u003ePrkcg\u003c/em\u003e, and \u003cem\u003eAtp2b2\u003c/em\u003e were related to postsynaptic cytosol and platelet calcium homeostasis, and \u003cem\u003eArc\u003c/em\u003e and \u003cem\u003eDlg4\u003c/em\u003e were related to the regulation of synaptic plasticity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Further, the down-regulated key candidates such as \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl12\u003c/em\u003e, \u003cem\u003eCcl7\u003c/em\u003e, were related to the regulation of natural killer cell chemotaxis, and \u003cem\u003eNupr1\u003c/em\u003e, \u003cem\u003eSlc38a3\u003c/em\u003e, \u003cem\u003eIfi209\u003c/em\u003e, \u003cem\u003eSfrp1\u003c/em\u003e were related to the inhibition of SOCS3-mediated cell signaling and participate in the inhibition of inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eT. gondii\u003c/em\u003e, a typical representative manipulator parasite [34, 35], can significantly affect the psychological behavior of the host and thus threaten public health [36, 37, 38]. It has been proved that neurogliocytes could be continuously activated by \u003cem\u003eT. gondii\u003c/em\u003e infection, and the infected mice could present a marked decrease in microglia population compared to non-infected group, both with the ME49 clonal strain and \u003cem\u003eTgCkBrRN2\u003c/em\u003e atypical strain (CK2) [39]. Therefore, we have established \u003cem\u003ein vitro\u003c/em\u003e infection models of Wh6 and LHG strains by using BV2 cell line, to better understand their invasion ability to microglia. Our current results demonstrated that Wh6 has a significantly more intensive ability than LHG in terms of microglia invasion and intracellular proliferation \u003cem\u003ein vitro\u003c/em\u003e. Consistently, it has been proved that in the mice infected with \u003cem\u003eTgCtWH6\u003c/em\u003e strain, the hosts appeared apparently cognitive behavioral disorders, and the neuronal apoptosis, Aβ deposition, and neuroinflammatory response involved in microglia polarization may be the molecular and cellular mechanisms [40]. We have also found that the Wh6-infected mice showed significant cognitive impairment, speculating that the increased cognitive impairment caused by WH6 may be related to its stronger ability to microglia invasion. Moreover, our results also found that LHG had a stronger brain cyst formation ability in the infected mice, as well as an increased anxiety-like behavioral disorder during the chronic infection phase. Thus, our results further remind us that there may be a direct correlation between the brain cyst formation ability and the degree of behavioral abnormalities.\u003c/p\u003e \u003cp\u003eSimultaneously, our ONT RNA-seq results revealed that the significantly up-regulated DETs in the two infected groups were primarily related to immune response and pathogen infection defense-related pathways, also consistent with the previous studies [41]. However, these studies mainly focused on the intensive virulent type I strain (\u003cem\u003eeg\u003c/em\u003e. RH) and the moderately virulent type II strains (\u003cem\u003eeg\u003c/em\u003e. Prugniaud and ME49) [42, 43, 44]. We believe our results would provide a necessary supplement for better understanding the mechanism of hosts' mental and behavioral disorders manipulated by \u003cem\u003eT. gondii\u003c/em\u003e strains prevalent in China with different genotypes.\u003c/p\u003e \u003cp\u003eImportantly, we also found that \u003cem\u003eT. gondii\u003c/em\u003e strain with different genotypes could regulate host neuroinflammatory responses through different pathways. For instance, Wh6 and LHG may mediate the host neuroinflammatory response by up-regulating the differential MHC class molecules. Further, our results showed that genes related to synaptic transmission function were primarily down-regulated by Wh6 whereas genes related to neurodegenerative diseases were significantly down-regulated by LHG. In addition, we found that the mortality of LHG-infected mice had a significant gender preference in the acute infection stage. As a matter of fact, it has been indicated that the mortality of female mice infected with \u003cem\u003eT. gondii\u003c/em\u003e was generally higher than that of male mice, and the reason may be the hypothesis that female mice could produce more inflammatory cytokines in response to \u003cem\u003eT. gondii\u003c/em\u003e infection [45]. Our results precisely indicated that in the TgW vs TgL comparable group, the enrichment of response to IL-8 in the top 10 GO analysis in TgL group was more significant, indicating that the previous hypothesis is reasonable.\u003c/p\u003e \u003cp\u003eAs to the selected key candidates in the present study, the significant DETs in the different infected groups could also reflect the differences in the manipulation mechanism. For example, genes associated with positive regulation of monocyte chemotaxis, such as \u003cem\u003eFcgr4\u003c/em\u003e, \u003cem\u003eH2-Aa\u003c/em\u003e, \u003cem\u003eH2-Ab1\u003c/em\u003e, and \u003cem\u003eCd74\u003c/em\u003e, were up-regulated in the TgW group, while genes associated with positive regulation of T cell chemotaxis, such as \u003cem\u003eH2-Q7\u003c/em\u003e and \u003cem\u003eH2-D1\u003c/em\u003e, were up-regulated in the TgL group. Since \u003cem\u003eFcgr4\u003c/em\u003e, low-affinity immunoglobulin gamma Fc region receptor IV, has been reported to be up-regulated in \u003cem\u003eleishmaniasis-\u003c/em\u003einfected mice, and ligation of FCGR4 by antigen-IgE(b) immune complexes could promote macrophage-mediated phagocytosis, antigen presentation to T cells, and proinflammatory cytokine production [46], so it has also been used to be a potential diagnostic marker for cutaneous leishmaniasis [47]. Secondly, the up-regulated \u003cem\u003eH2-Q7\u003c/em\u003e (i.e., H-2 class I histocompatibility antigen, Q7 alpha chain) belongs to the adaptive immune MHC I family response class, and it has been proved to participate in foreign antigen presentation, endogenous processing, and presentation of process polypeptide antigens, thus could promote the production of immune factors by T cells, and then enhance adaptive immunity [48]. These key candidates indicate that Wh6 strain was more likely to stimulate the phagocytosis and killing of the host central immune cells, whereas the LHG strain was more immunogenic to the host central immune system. This may be responsible for comprehending why LHG has a higher ability for brain cyst formation. Besides, there are several previous studies showed that WH6 strain could cause neuroinflammation, synaptic loss, and cognitive deficits in C57BL/6J mice, and the behavioral disorders phenotype were perfectly consistent with our study [49, 50] Simultaneously, in Tao' study, the tachyzoites of WH6 have been verified that they could indirectly induce APP protein production in HT22 by polarized BV2 cells, and the expression of CD80, pro-inflammatory factors, notch, and hes1 could be enhanced in the infected BV2 [49]. Moreover, the RNA-seq results in Wu' study [51] have also indicated that the WH6 infection could trigger extensive neuroinflammation in the prefrontal cortex, and the related genes such as \u003cem\u003eFcgr4\u003c/em\u003e, \u003cem\u003eCcl22\u003c/em\u003e and \u003cem\u003eH2-D1\u003c/em\u003e were highly consistent with our present research.\u003c/p\u003e \u003cp\u003eAs to the down-regulated key candidates, genes related to synaptic transmissions such as \u003cem\u003eUnc13a\u003c/em\u003e, \u003cem\u003ePrkcg\u003c/em\u003e, \u003cem\u003eSept4\u003c/em\u003e, and \u003cem\u003eKcng4\u003c/em\u003e have been selected from the TgW group, while genes related to neurodegeneration such as \u003cem\u003eCrh\u003c/em\u003e and \u003cem\u003eNdrg2\u003c/em\u003e were selected from the TgL group. Firstly, it is well known that the \u003cem\u003eUnc13\u003c/em\u003e (Protein unc-13 homolog C) family is critical in regulating neurotransmitter release and synaptic plasticity in CNS. \u003cem\u003eUnc13a\u003c/em\u003e is involved in several related processes including glutamatergic synaptic transmission, synaptic vesicle maturation, and the regulation of synaptic transmission at parallel fiber-Purkinje cell synapses [52]. Secondly, \u003cem\u003eNdrg2\u003c/em\u003e is an \u003cem\u003eN\u003c/em\u003e-myc downstream regulatory gene family member. It is primarily expressed in the astrocytes and involved in the pathogenesis of many neurological diseases, such as stroke, neurodegenerative diseases (Alzheimer's and Parkinson's diseases), and psychiatric disorders (depression and attention deficit hyperactivity disorder) [53]. This would be auxiliary to explain why the infected animals showed a loss of natural fear of predators, anxiety behaviors, and memory impairments.\u003c/p\u003e \u003cp\u003eImportantly, expression of genes related to synaptic plasticity and neurological diseases such as \u003cem\u003eArc\u003c/em\u003e, \u003cem\u003eHomer1\u003c/em\u003e, and \u003cem\u003eItpr1\u003c/em\u003e in the TgL group were significantly up-regulated compared to the TgW group. Arc is an activity-regulated cytoskeleton-associated protein. It can regulate synaptic strength through multiple mechanisms and is essential for memory consolidation and learning [54]. In the meanwhile, the expression of natural killer cell chemotaxis-related genes such as \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl12\u003c/em\u003e, \u003cem\u003eH2-Q4, and Slc38a3\u003c/em\u003e in the TgL group was lower expressed than that of the TgW group. Particularly, CCL2, a ligand for C-C chemokine receptor CCR2, could act on dopaminergic neurons to increase their excitability, dopamine release, and motor activity, as well as increase NMDA-mediated synaptic transmission in neurons containing dopamine D1 and D2 receptors [55]. As a result, our founding may provide a further understanding of why LHG could induce more severe anxiety-like behaviors.\u003c/p\u003e \u003cp\u003eMental and behavioral manipulation mechanisms of difference \u003cem\u003eT. gondii\u003c/em\u003e strains to their host are complex. Although our results promote a hypothesis that difference of neuroinflammatory response and synaptic transmission may be the key pathways for the difference of invasion and mental manipulation ability between the two \u003cem\u003eT. gondii\u003c/em\u003e strains. However, the accurate molecular mechanisms need to be further verified.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, both \u003cem\u003eChinese I\u003c/em\u003e Wh6 strain and \u003cem\u003eChinese III\u003c/em\u003e LHG strain could cause cognitive and spatial memory impairment, and anxiety-like behaviors in the hosts, but the anxiety caused by LHG appeared more obvious. Immune response and synaptic transmission were significantly enriched and may be the major influenced pathways. Besides, our results indicated that genes related to synaptic transmission such as \u003cem\u003eUnc13c, Prkcg, Sept4, and Kcng4\u003c/em\u003e may be the main factors responsible for Wh6 mental and behavioral manipulation; while genes related to neurodegeneration such as \u003cem\u003eNdrg2\u003c/em\u003e and \u003cem\u003eArc\u003c/em\u003e, as well as \u003cem\u003eCcl2\u003c/em\u003e, maybe the key factors for explaining the stronger mental manipulation ability of LHG strain (Graphical abstract).\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eT. gondii\u003c/em\u003e: \u003cem\u003eToxoplasma gondii\u003c/em\u003e; Wh6: Chinese cat-derived type I \u003cem\u003eTgCtWh6\u003c/em\u003e strain of \u003cem\u003eT. gondii\u003c/em\u003e; LHG: human type III \u003cem\u003eTgCtLHG\u003c/em\u003e strain of \u003cem\u003eT. gondii\u003c/em\u003e; DETs: differentially expressed transcripts; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: protein\u0026ndash;protein interaction; qPCR: quantitative polymerase chain reaction; AIDS: Acquired Immunodeficiency Syndrome; CNS: Central Nervous System; DMEM: Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium; FBS: fetal bovine serum; Con: control group; TgW: Wh6-infected; TgL: LHG-infected; OFT: open-field: test; YM: Y-maze; PBS: sterile phosphate-buffered saline; SEMs: standard errors of the means; ONT: Oxford Nanopore Technologies; BPs: biological processes; EB: Epstein\u0026ndash;Barr; GABA: Gamma-aminobutyric acid; HIV: Human Immunodeficiency Virus; AMPA: \u0026alpha;-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid; cGMP-PKG: cyclic guanosine monophosphate-protein kinase G; MHC: major histocompatibility complex; TAP1: Transporter associated with antigen processing 1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at: xxxDeclarations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals (issued by the Council for the International Organizations of Medical Sciences), as well as the guidelines set by the Institutional Animal Care and Use Committee of Shandong First Medical University (approval number: w202103030088) and by Animal Research: Reporting of \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eExperiments (ARRIVE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e’s\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKY, GHZ and JPC conceived the study, designed the experiments and critically revised the manuscript. BBZ, HJD, HS and XXM performed the experiments, analyzed the data and drafted the manuscript. HHX, WJZ, YNL and CX participated in the implementation of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Parasitic Pathogens of National Health Committee and Vector Biology Key Laboratory Open Research Topic [grant number NHCKFKT2022-15]; the Natural Science Foundation of Shandong Province [grant number ZR2022MH197]; the Medical and Health Science and Technology Program of Shandong Province [grant number 202401050156]; and the Taishan Scholars Project of Shandong Province [grant number tsqn202103186].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are included in the article and its Supplementary Material. Further any further inquiries, please contact the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Prof. Jilong Shen (Anhui Medical University, Hefei, China) for generously providing the \u003cem\u003eChinese I\u003c/em\u003e \u003cem\u003eT. gondii\u003c/em\u003e strain-\u003cem\u003eTgCtwh6\u003c/em\u003e and the \u003cem\u003eChinese III\u003c/em\u003e \u003cem\u003eT. gondii\u003c/em\u003e strain-\u003cem\u003eTgCtLHG\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology, Shanghai 200025, China;\u0026nbsp;Shandong Institute of Parasitic Diseases, Jining 272033, People's Republic of China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eKey Laboratory of Parasite and Vector Biology, National Health Commission of the People's Republic of China, Shanghai 200025, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTenter AM, Heckeroth AR, Weiss LM. \u003cem\u003eToxoplasma gondii\u003c/em\u003e: from animals to humans. 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J Exp Biol. 2013;216 Pt 1:99\u0026ndash;112.\u003c/li\u003e\n\u003cli\u003eBrito RMM, da Silva MCM, Vieira-Santos F, de Almeida Lopes C, Souza JLN, Bastilho AL, et al. Chronic infection by atypical \u003cem\u003eToxoplasma gondii\u003c/em\u003e strain induces disturbance in microglia population and altered behaviour in mice. Brain Behav Immun Health. 2023;30:100652.\u003c/li\u003e\n\u003cli\u003eXu D, Yan Z, Zhou Y, He Y, Liu S, Gao Z, et al. beta-Glucan ameliorates anxiety-like behavior in mice chronically infected with the \u003cem\u003eToxoplasma gondii\u003c/em\u003e Wh6 strain. Parasitol Res. 2022;121 12:3513-21.\u003c/li\u003e\n\u003cli\u003eHu RS, He JJ, Elsheikha HM, Zou Y, Ehsan M, Ma QN, et al. Transcriptomic Profiling of Mouse Brain During Acute and Chronic Infections by \u003cem\u003eToxoplasma gondii\u003c/em\u003e Oocysts. Front Microbiol. 2020;11:570903.\u003c/li\u003e\n\u003cli\u003eJia B, Lu H, Liu Q, Yin J, Jiang N, Chen Q. 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Semin Cell Dev Biol. 2018;77:10\u0026thinsp;\u0026minus;\u0026thinsp;6.\u003c/li\u003e\n\u003cli\u003eGuyon A, Skrzydelski D, De Giry I, Rovere C, Conductier G, Trocello JM, et al. Long term exposure to the chemokine CCL2 activates the nigrostriatal dopamine system: a novel mechanism for the control of dopamine release. Neuroscience. 2009;162 4:1072-80.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Toxoplasma gondii, Cerebral cysts, Differentially expressed transcripts, Nanopore RNA-seq, Mental and behavioral disorders","lastPublishedDoi":"10.21203/rs.3.rs-6075517/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6075517/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eToxoplasma gondii\u003c/em\u003e (\u003cem\u003eT. gondii\u003c/em\u003e) can cause severe damage to immunodeficient hosts, and also compromise brain structure and function in immunocompetent hosts during latent infection. In China, the two different isolates, \u003cem\u003eChinese I\u003c/em\u003e (\u003cem\u003eToxoDB#9\u003c/em\u003e) and \u003cem\u003eChinese III\u003c/em\u003e are dominant epidemic strains widely spreading in humans and domestic animals and can lead to latent infection in host brain tissues, but the comparison of their manipulation patterns and mechanisms remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTachyzoites of \u003cem\u003eTgWh6\u003c/em\u003e (Wh6) strain and \u003cem\u003eTgCtLHG\u003c/em\u003e (LHG) strain were used for establishing \u003cem\u003ein vitro\u003c/em\u003e infection models within mouse microglia BV2 cells, and the differences in their invasion and proliferation patterns were observed. C57BL/6J mice were used to establish \u003cem\u003ein vivo\u003c/em\u003e latent infection models. After behavioral tests, the differential expressed transcripts (DETs) of the infected and control animals' cerebral cortex were sequenced by Nanopore RNA-seq.\u0026nbsp;Functional differences of DETs were analyzed by Gene Ontology enrichment analysis (GO), Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG), and protein-protein interaction (PPI) and cluster analysis. Expression of the key candidates were verified by quantitative polymerase chain reaction (qPCR).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn our infection models, we found that Wh6 had more vigorous invasion and proliferation abilities \u003cem\u003ein vitro\u003c/em\u003e, while LHG had greater ability to form cysts \u003cem\u003ein vivo\u003c/em\u003e. In the latent infection phase, behavioral changes including spatial working memory, cognitive and motor abilities, and anxiety were apparently observed in both Wh6 and LHG infected mice, however, the LHG group showed more serious anxiety. Among DETs, genes related to MHC class II molecules were significantly up-regulated both in the infected mice, while genes related to synaptic transmission and neurodegenerative diseases were respectively down-regulated in the infected groups. The downregulated DETs of \u003cem\u003eSept4\u003c/em\u003e, \u003cem\u003eKcng4\u003c/em\u003e, \u003cem\u003eUnc13c\u003c/em\u003e, and \u003cem\u003ePrkcg\u003c/em\u003e in the WH6 group, which are related to synaptic transmission; and \u003cem\u003eNdrg2\u003c/em\u003e and \u003cem\u003eArc\u003c/em\u003e in the LHG group, which are related to neurodegenerative diseases, would be selected to be the key candidates in the latent infection phase.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCompared with WH6, although LHG has a milder invasion ability, it can cause increased behavioral disorders in hosts. Genes related to synaptic transmission and neurodegenerative diseases may be the main causes of host mental and behavioral disorders.\u003c/p\u003e","manuscriptTitle":"Effects of latent infection of Toxoplasma gondii strains with different genotypes on mouse behavior and brain transcripts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 20:11:17","doi":"10.21203/rs.3.rs-6075517/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-28T00:15:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-27T23:07:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-27T08:04:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149102032084789660861735673874244647755","date":"2025-04-19T23:55:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T23:52:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-17T10:21:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-04-17T03:05:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6d47883-d22e-41ef-902c-762b23ed8c2c","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:08:26+00:00","versionOfRecord":{"articleIdentity":"rs-6075517","link":"https://doi.org/10.1186/s13071-025-06819-7","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-05-26 15:57:35","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2025-04-21 20:11:17","video":"","vorDoi":"10.1186/s13071-025-06819-7","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06819-7","workflowStages":[]},"version":"v1","identity":"rs-6075517","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6075517","identity":"rs-6075517","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}