The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

The antioxidant protein glutaredoxin 1 is essential for oxidative stress response and pathogenicity of Toxoplasma gondii.

Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs in the pathogenicity of Toxoplasma gondii. We show that Grxs are localized in the mitochondria (Grx1), cytoplasm (Grx2), and apicoplast (Grx3, Grx4), while Grx5 had an undetectable level of expression. We generated Δgrx1-5 mutants of T. gondii type I RH and type II Pru strains using CRISPR-Cas9 system. No significant differences in the infectivity were detected between four Δgrx (grx2-grx5) strains and their respective wild-type (WT) strains in vitro or in vivo. Additionally, no differences were detected in the production of reactive oxygen species, total antioxidant capacity, superoxide dismutase activity, and sensitivity to external oxidative stimuli. Interestingly, RHΔgrx1 or PruΔgrx1 exhibited significant differences in all the investigated aspects compared to the other grx2-grx5 mutant and WT strains. Transcriptome analysis suggests that deletion of grx1 altered the expression of genes involved in transport and metabolic pathways, signal transduction, translation, and obsolete oxidation-reduction process. The data support the conclusion that grx1 supports T. gondii resistance to oxidative killing and is essential for the parasite growth in cultured cells and pathogenicity in mice and that the active site CGFS motif was necessary for Grx1 activity.

The protein phosphatase 2A holoenzyme is a key regulator of starch metabolism and bradyzoite differentiation in Toxoplasma gondii.

Phenotypic switching between tachyzoite and bradyzoite is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Although accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage, the regulatory factors and mechanisms contributing to amylopectin storage in bradyzoites are incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit PP2A-C, a scaffold subunit PP2A-A and a regulatory subunit PP2A-B. Disruption of any of these subunits increased starch accumulation and blocked the tachyzoite-to-bradyzoite differentiation. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Phosphoproteomics identified several putative PP2A holoenzyme substrates that are involved in bradyzoite differentiation. Our findings provide novel insight into the role of PP2A as a key regulator of starch metabolism and bradyzoite differentiation in T. gondii.

Seroprevalence and risk factors of Toxoplasma gondii and Neospora caninum infection in black goats in Yunnan Province, Southwestern China.

Toxoplasma gondii and Neospora caninum are two obligate intracellular protozoan parasites that can cause reproductive failure and production losses. To date, there is no data of T. gondii and N. caninum seroprevalence in black goats in Yunnan Province, southwestern China. In the present study, a total of 734 serum samples were collected from black goats in four different counties of Yunnan Province. 734 and 590 serum samples were examined for antibodies against T. gondii and N. caninum by using MAT and indirect ELISA, respectively. A total of 123 and 76 samples were T. gondii-positive and N. caninum-positive, respectively. The overall seroprevalence of T. gondii in black goats was 16.76% (123/734, 95% CI: 14.06-19.46) with the titer ranged from 1:25 to 1:3200. The seroprevalence of N. caninum was 12.88% (76/590, 95% CI: 10.18-15.58). There was significant difference in seroprevalence of N. caninum in different regions (P < 0.01, χ2 = 30.63) and age groups (P < 0.05, χ2 = 11.85). Significant differences in seroprevalence of T. gondii were observed in different regions (P < 0.05, χ2 = 9.21) and different gender groups (P < 0.01, χ2 = 12.29). Results of seroprevalence of T. gondii and N. caninum indicated that T. gondii and N. caninum were prevalent parasites in black goats in Yunnan Province. This is the first report of seroprevalence of T. gondii and N. caninum in black goats in Yunnan Province. The results of this study indicated that some measures should be taken to control these two parasites and to reduce economic losses to the livestock industry in Yunnan Province.

Global profiling of protein lysine malonylation in Toxoplasma gondii strains of different virulence and genetic backgrounds.

Lysine malonylation is a post-translational modification (PTM), which regulates many cellular processes. Limited information is available about the level of lysine malonylation variations between Toxoplasma gondii strains of distinct genetic lineages. Yet, insights into such variations are needed to understand the extent to which lysine malonylation contributes to the differences in the virulence and repertoire of virulence factors between T. gondii genotypes. In this study, we profiled lysine malonylation in T. gondii using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification. This analysis was performed on three T. gondii strains with distinctive pathogenicity in mice, including RH strain (type I), PRU strain (type II), and VEG strain (type III). In total, 111 differentially malonylated proteins and 152 sites were upregulated, and 17 proteins and 17 sites were downregulated in RH strain versus PRU strain; 50 proteins and 59 sites were upregulated, 50 proteins and 53 sites were downregulated in RH strain versus VEG strain; and 72 proteins and 90 sites were upregulated, and 7 proteins and 8 sites were downregulated in VEG strain versus PRU strain. Differentially malonylated proteins were involved in key processes, such as those mediating the regulation of protein metabolism, stress response, glycolysis, and actin cytoskeleton. These results reveal an association between lysine malonylation and intra-species virulence differences in T. gondii and offer a new resource for elucidating the contribution of lysine malonylation to energy metabolism and virulence in T. gondii.

The Role of Type II Fatty Acid Synthesis Enzymes FabZ, ODSCI, and ODSCII in the Pathogenesis of Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI, and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔFabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔODSCI and RHΔODSCII mutant strains no similar changes were detected. RHΔFabZ exhibited reduced virulence for mice compared with RHΔODSCI and RHΔODSCII, as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔODSCI and PRUΔODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii, while ODSCI and ODSCII do not contribute to these traits.

Characterization of functions in parasite growth and virulence of four Toxoplasma gondii genes involved in lipid synthesis by CRISPR-Cas9 system.

Fatty acid uptake is extremely important for the survival and growth of the intracellular parasite Toxoplasma gondii. In this study, CRISPR-Cas9 gene editing technology was used to investigate the role of four lipid synthesis enzymes, namely, glycerol-3-phosphate dehydrogenase (G3PDH), malonyl CoA-acyl carrier protein transacylase (FabD), acyl-ACP thiolesterase (TE), and diacylglycerol acyltransferase (DGAT), in the virulence and infectivity of Type I RH and Type II Prugniaud (Pru) strains of T. gondii. Immunofluorescence analysis of the tachyzoite stage showed that FabD protein was located in the apicoplast; however, the expression level of the other three proteins was undetectable. Compared with wild-type (WT) strains, the growth of RHΔG3PDH, RHΔTE, and RHΔDGAT in vitro and their virulence in vivo were not significantly different. However, RHΔFabD exhibited a significantly reduced growth rate, compared with the WT strain. The deletion of FabD attenuated the virulence of Type II Pru strain and reduced the formation of cysts in vivo. These data improved our understanding of the role of lipid synthesis enzymes in the pathogenesis of T. gondii.

Functional Characterization of 17 Protein Serine/Threonine Phosphatases in Toxoplasma gondii Using CRISPR-Cas9 System.

Protein serine/threonine phosphatases (PSPs), found in various plants and protozoa, are involved in the regulation of various biological processes. However, very little is known about the role of PSPs in the pathogenicity of the apicomplexan protozoan Toxoplasma gondii. Herein, the subcellular localization of 17 PSPs (PP5, PP7, EFPP, SLP, PPM3F, PPM4, PPM5A, PPM5B, PPM6, PPM8, PPM9, PPM12, PPM14, PPM18, CTD1, CTD2, and CTD3) was examined by 6× HA tagging of endogenous genes in C-terminal. The PSPs were detected in the cytoplasm (PP5, EFPP, PPM8, and CTD2), dense granules (SLP), nucleus (PPM4 and PPM9), inner membrane complex (PPM12), basal complex (CTD3), and apical pole (PP7). The remaining PSPs exhibited low or undetectable level of expression. To characterize the contribution of these genes to the infectivity of T. gondii, knock-out (KO) strains of type I RH strain deficient in the 17 psp genes and KO type II Pru strain deficient in pp7 and slp genes were constructed. The pathogenicity of individual RHΔpsp mutants was characterized in vitro using plaque, egress, and intracellular replication assays, and mouse infection, while pathogenicity of PruΔpp7 and PruΔslp mutant strains was evaluated by examining the parasite lytic cycle in vitro and assessment of brain cyst burden in mice. No significant differences were observed between 16 RHΔpsp strains and wild-type (WT) RH strain. However, RHΔpp7 exhibited significantly lower invasion efficiency and parasitophorous vacuole formation in vitro, and less virulence in mice compared with other RHΔpsp and WT strains. In addition, PruΔpp7 exhibited marked attenuation of virulence and significant reduction in the brain cyst burden in mice compared with PruΔslp and WT strains, suggesting the key role of PP7 in the virulence of T. gondii. Comparative transcriptomic profiling of the 17 psp genes showed that they may play different roles in the pathogenesis of different genotypes or life cycle stages of T. gondii. These findings provide new insight into the role of PSPs in the pathogenesis of T. gondii.

Global profiling of lysine 2-hydroxyisobutyrylome in Toxoplasma gondii using affinity purification mass spectrometry.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that Khib plays roles in the activity of gene transcription and Khib-containing proteins are closely related to the cellular metabolism. In this study, a global Khib-containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 Khib modification sites across 400 Khib-containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that Khib-modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be Khib-modified, suggesting the involvement of Khib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had Khib sites. These results expanded our understanding of the roles of Khib in T. gondii and should promote further investigations of how Khib regulates gene expression and key biological functions in T. gondii.

Advances in the Development of Anti-Haemonchus contortus Vaccines: Challenges, Opportunities, and Perspectives.

The gastrointestinal nematode parasite Haemonchus contortus (H. contortus) is a resident of tropical and subtropical regions worldwide that imposes significant production losses, economic losses, and animal health issues in the small ruminant industry, particularly sheep and goats. Considerable efforts have been made to understand how immunity is elicited against H. contortus infection. Various potential vaccine antigens have been tested by different methods and strategies applied in animal models, and significant progress has been made in the development of vaccines against H. contortus. This review highlighted and shared the knowledge about the current understanding of host immune responses to H. contortus and ongoing challenges in the development of a protective, effective, and long-lasting vaccine against H. contortus infection. We have also pinpointed some achievements and failures in the development and testing of vaccines, which will establish a road map for future research directions to explore new effective vaccine candidates for controlling and preventing H. contortus infection.

First Report of Chlamydia Seroprevalence and Risk Factors in Domestic Black-Boned Sheep and Goats in China.

The Gram-negative bacteria of the genus Chlamydia cause a wide range of diseases in humans and animals. The seroprevalence of Chlamydia in domestic black-boned sheep and goats in China is unknown. In this survey, a total of 481 serum samples were collected randomly from domestic black-boned sheep and goats from three counties in Yunnan province, southwest China, from July to August 2017. The sera were examined by an indirect hemagglutination assay (IHA). Antibodies to Chlamydia were detected in 100/481 [20.79%, 95% confidence interval (CI), 17.16-24.42] serum samples (IHA titer ≥1:64). The Chlamydia seroprevalence ranged from 12.21% (95% CI, 7.81-16.61) to 30.89% (95% CI, 22.72-39.06) across different regions in Yunnan province, and the differences were statistically significant (P < 0.01). The seroprevalence in male domestic black-boned sheep and goats (28.64%; 95% CI, 22.36-34.92) was significantly higher than that in the females (15.25%; 95% CI, 11.05-19.45) (P < 0.01). However, there was no statistically significant difference in Chlamydia seroprevalence in domestic black-boned sheep and goats between ages and species (P > 0.05). To our knowledge, this is the first report of Chlamydia seroprevalence in domestic black-boned sheep and goats in Yunnan Province, southwest China. These data provide baseline information for future implementation of measures to control Chlamydia infection in these animals.

Effect of deletion of gra17 and gra23 genes on the growth, virulence, and immunogenicity of type II Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection.

Serological evidence of Toxoplasma gondii and Chlamydia infection in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Toxoplasma gondii, Neospora caninum, Chlamydia and bluetongue virus (BTV) are four important pathogens which can cause reproductive loss. The present study was conducted to estimate the seroprevalence of T. gondii, N. caninum, Chlamydia and BTV in alpacas in Shanxi Province, northern China. A total of 251 serum samples were collected from alpacas, and antibodies against T. gondii and Chlamydia were examined by the modified agglutination test (MAT) and indirect hemagglutination assay (IHA), respectively. Antibodies to N. caninum and BTV were determined by using the commercially available competitive enzyme-linked immunosorbent assay (cELISA) kits, respectively. The overall seroprevalence of T. gondii was 9.16% (95% CI 5.59-12.73) in the three sampled counties, of which, no T. gondii-seropositive samples were detected in alpacas in Fanshi County. Gender differences in the T. gondii seroprevalence were observed. The overall Chlamydia seroprevalence was 13.94% (95% CI: 9.66-18.22), and there was a statistically significant difference in Chlamydia seroprevalence in alpacas between the two counties, Jiexiu and Fanshi. All serum samples tested negative for N. caninum and BTV antibodies, respectively. To our knowledge, this is the first report of T. gondii and Chlamydia seroprevalence in alpacas in China, which provides baseline information for controlling T. gondii and Chlamydia infection in alpacas in China.

Global Proteomic Analysis of Lysine Malonylation in Toxoplasma gondii.

Lysine malonylation (Kmal) is a new post-translational modification (PTM), which has been reported in several prokaryotic and eukaryotic species. Although Kmal can regulate many and diverse biological processes in various organisms, knowledge about this important PTM in the apicomplexan parasite Toxoplasma gondii is limited. In this study, we performed the first global profiling of malonylated proteins in T. gondii tachyzoites using affinity enrichment and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Three experiments performed in tandem revealed 294, 345, 352 Kmal sites on 203, 236, 230 malonylated proteins, respectively. Computational analysis showed the identified malonylated proteins to be localized in various subcellular compartments and involved in many cellular functions, particularly mitochondrial function. Additionally, one conserved Kmal motif with a strong bias for cysteine was detected. Taken together, these findings provide the first report of Kmal profile in T. gondii and should be an important resource for studying the physiological roles of Kmal in this parasite.

Prevalence and multilocus genotyping of Giardia duodenalis in Tan sheep (Ovis aries) in northwestern China.

Giardia duodenalis is a common intestinal protozoa, which can cause the occurrence of diarrhea, weight loss, and even death in animals or human, this threatens the husbandry industry and public health. It can infect virtually humans and all domestic animals including sheep. Tan sheep is one of the most important sheep breeds, which is short-tailed indigenous sheep breed used for production of high quality meat and pelts in China. However, there are no report regarding the occurrence and multilocus genotyping of G. duodenalis in Tan sheep in northwestern China. Thus, the objective of the present study was to investigate the prevalence and multilocus genotypes of G. duodenalis in Tan sheep. 1014 fecal samples were collected from Tan sheep from Ningxia Hui Autonomous Region, and three loci (ß-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes) were amplified by nested PCR. The prevalence of G. duodenalis in Tan sheep was 14.5% (147/1014), two assemblages (assemblage A, n = 43; and E, n = 90) were detected, including one novel assemblage A at bg locus, one novel assemblage A at tpi locus, and 10 and 11 novel subtypes of assemblage E were detected at the bg and gdh loci, respectively. One MLGs was formed based on sequence variation among the three loci. Moreover, 9 Tan sheep were infected with two assemblages (A and E) based on the three loci. These findings expand the host range of G. duodenalis and revealed genetic diversity of G. duodenalis assemblages in Tan sheep.

RHΔgra17Δnpt1 Strain of Toxoplasma gondii Elicits Protective Immunity Against Acute, Chronic and Congenital Toxoplasmosis in Mice.

: In the present study, a dense granule protein 17 (gra17) and novel putative transporter (npt1) double deletion mutant of Toxoplasma gondii RH strain was engineered. The protective efficacy of vaccination using RHΔgra17Δnpt1 tachyzoites against acute, chronic, and congenital toxoplasmosis was studied in a mouse model. Immunization using RHΔgra17Δnpt1 induced a strong humoral and cellular response, as indicated by the increased levels of anti-T. gondii specific IgG, interleukin 2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-γ). Vaccinated mice were protected against a lethal challenge dose (103 tachyzoites) of wild-type homologous (RH) strain and heterologous (PYS and TgC7) strains, as well as against 100 tissue cysts or oocysts of Pru strain. Vaccination also conferred protection against chronic infection with 10 tissue cysts or oocysts of Pru strain, where the numbers of brain cysts in the vaccinated mice were significantly reduced compared to those detected in the control (unvaccinated + infected) mice. In addition, vaccination protected against congenital infection with 10 T. gondii Pru oocysts (administered orally on day 5 of gestation) as shown by the increased litter size, survival rate and the bodyweight of pups born to vaccinated dams compared to those born to unvaccinated + infected dams. The brain cyst burden of vaccinated dams was significantly lower than that of unvaccinated dams infected with oocysts. Our data show that T. gondii RHΔgra17Δnpt1 mutant strain can protect mice against acute, chronic, and congenital toxoplasmosis by balancing inflammatory response with immunogenicity.

Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice.

In this study, we generated a tkl1 deletion mutant in the Toxoplasma gondii type 1 RH (RHΔtkl1) strain and tested the protective efficacies of vaccination using RHΔtkl1 tachyzoites against acute, chronic, and congenital T. gondii infections in Kunming mice. Mice vaccinated with RHΔtkl1 mounted a strong humoral and cellular response as shown by elevated levels of anti-T. gondii-specific IgG, IL-2, IL-12, IFN-γ, and IL-10. All RHΔtkl1-vaccinated mice survived a lethal challenge with 1 × 103 tachyzoites of type 1 RH or ToxoDB#9 (PYS or TgC7) strain as well as 100 cysts or oocysts of Prugniuad strain. All mock-vaccinated plus infected mice have died. Vaccination also protected against cyst- or oocyst-caused chronic infection, reduced vertical transmission caused by oocysts, increased litter size, and maintained body weight of pups born to dams challenged with 10 oocysts on day 5 of gestation. In contrast, all mock-vaccinated plus oocysts-infected dams had aborted, and no fetus has survived. Vaccinated dams remained healthy postinfection, and their brain cyst burden was significantly reduced compared with mock-vaccinated dams infected with oocysts. In vivo depletion of CD4+ T cells, CD8+ T cells, and B cells revealed that CD8+ T cells are involved in the protection of mice against T. gondii infection. Additionally, adoptive transfer of CD8+ T cells from RHΔtkl1-vaccinated mice significantly enhanced the survival of naive mice infected with the pathogenic strain. Together, these data reaffirm the importance of CD8+ T cell responses in future vaccine design for toxoplasmosis and present T. gondii tkl1 gene as a promising vaccine candidate.

Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection.

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice.

Serological evidence of Toxoplasma gondii and Neospora caninum infection in black-boned sheep and goats in southwest China.

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49-41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02-11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ2 = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ2 = 8.558, df = 2, P<0.05), age (χ2 = 16.631, df = 3, P < 0.01), gender (χ2 = 11.219, df = 1, P < 0.01) and species (χ2 = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94-5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats.

Prevalence and subtypes of Blastocystis sp. infection in zoo animals in three cities in China.

Blastocystis is a highly prevalent eukaryotic parasite of many animals and humans worldwide. It can compromise the gastrointestinal tract and cause gastrointestinal symptoms, constituting a serious threat to human health and animal growth. Many animals are potential sources of Blastocystis infection in humans. However, limited data are available regarding the prevalence and subtype distribution of Blastocystis infection among zoo animals in China. Therefore, the present study examined the prevalence and subtypes of Blastocystis in zoo animals in Hangzhou, Dalian, and Suzhou cities, China. Of 450 fecal samples from zoo animals, 27 (6.0%) were PCR-positive for Blastocystis, with 7.7% (8/104), 11.3% (7/62), 16.7% (3/18), 1.8% (2/114), 6.3% (1/16), 9.5% (2/21), and 3.6% (4/109) in artiodactyla, aves, rodentia, nonhuman primates, perissodactyla, marsupialia, and carnivora, respectively. Significant differences in the prevalence of Blastocystis were found among different animal groups (P < 0.05). Sequence analysis showed 7 known subtypes (ST2, ST4, ST5, ST7, ST8, ST10, and ST14) of Blastocystis in the present study, with ST10 (10/27) as the predominant subtype in all three of the examined zoos. To our knowledge, this is the first report of Blastocystis infection in Damaliscus dorcas, Cervus elaphus, Macropus rufogriseus, Grus japonensis, Trichoglossus haematodus, Panthera tigris ssp. tigris (white), Panthera tigris ssp. altaica, Lycaon pictus, Suricata suricatta, and Dolichotis patagonum in China. These results demonstrate the presence of Blastocystis infection in zoo animals and provided baseline data for preventing and controlling Blastocystis infection in zoo animals and humans in China.