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.

Global proteomic profiling of multiple organs of cats (Felis catus) and proteome-transcriptome correlation during acute Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is a protozoan parasite which can infect almost all warm-blooded animals and humans. Understanding the differential expression of proteins and transcripts associated with T. gondii infection in its definitive host (cat) may improve our knowledge of how the parasite manipulates the molecular microenvironment of its definitive host. The aim of this study was to explore the global proteomic alterations in the major organs of cats during acute T. gondii infection. METHODS: iTRAQ-based quantitative proteomic profiling was performed on six organs (brain, liver, lung, spleen, heart and small intestine) of cats on day 7 post-infection by cysts of T. gondii PRU strain (Genotype II). Mascot software was used to conduct the student's t-test. Proteins with P values < 0.05 and fold change > 1.2 or < 0.83 were considered as differentially expressed proteins (DEPs). RESULTS: A total of 32,657 proteins were identified in the six organs, including 2556 DEPs; of which 1325 were up-regulated and 1231 were down-regulated. The brain, liver, lung, spleen, heart and small intestine exhibited 125 DEPs, 463 DEPs, 255 DEPs, 283 DEPs, 855 DEPs and 575 DEPs, respectively. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of all proteins and DEPs in all organs showed that many proteins were enriched in binding, cell part, cell growth and death, signal transduction, translation, sorting and degradation, extracellular matrix remodeling, tryptophan catabolism, and immune system. Correlations between differentially expressed proteins and transcripts were detected in the liver (n = 19), small intestine (n = 17), heart (n = 9), lung (n = 9) and spleen (n = 3). CONCLUSIONS: The present study identified 2556 DEPs in six cat tissues on day 7 after infection by T. gondii PRU strain, and functional enrichment analyses showed that these DEPs were associated with various cellular and metabolic processes. These findings provide a solid base for further in-depth investigation of the complex proteotranscriptomic reprogramming that mediates the dynamic interplays between T. gondii and the different feline tissues.

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.

Prevalence of Toxoplasma gondii infection in chickens in China during 1993-2021: a systematic review and meta-analysis.

Toxoplasma gondii is an important intracellular parasitic protozoan with a variety of hosts, including chickens, which poses a potential threat to public health. However, little is known regarding overall T. gondii infection in chickens in China. Herein, the prevalence and risk factors associated with T. gondii infection in chickens in China were investigated using a meta-analysis. Forty studies regarding the prevalence of T. gondii in chickens in China from 1993 to 2021 were identified using five databases (PubMed, Science Direct, CNKI, Wang Fang, and VIP). Quantitative and potential sources were analyzed through subgroup analysis and meta-regression in R v3.5.2. The overall prevalence of T. gondii in chickens in China was 13.4% (95% confidence interval (CI): 10.9-16.0). In the region subgroup, the lowest prevalence was presented in Northwestern China (6.0%, 95% CI: 3.2-9.5; P < 0.001). Seasonally, T. gondii prevalence was the highest in spring (17.9%, 95% CI: 7.7-30.9; P = 0.007). Among detection methods, the prevalence in the ELISA subgroup was the highest (22.8%, 95% CI: 17.1-29.1; P < 0.001). According to the farming mode, the prevalence of T. gondii in free-range chickens (19.5%, 95% CI: 15.4-23.9) was significantly higher than that in chickens raised by intensive farming (7.4%, 95% CI: 5.1-10.2; P < 0.001). We also estimated the relationships between region, sampling year, chicken age, chicken application, gender, sample classification, study quality, and T. gondii prevalence in chickens in China. Our study showed that region, season, and farming model played important roles in T. gondii infection of chickens. Integrated control measures should be undertaken to reduce the losses caused by T. gondii infection to the chicken industry.

Meta-analysis of the prevalence of Echinococcus in dogs in China from 2010 to 2019.

BACKGROUND: Echinococcosis (canine Echinococcus disease) is a neglected tropical disease that causes serious public harm. Dogs, as a terminal host of Echinococcus spp., are a key part of the Echinococcus epidemic. Echinococcosis spreads easily in humans and animals in some areas of China and it is therefore necessary to fully understand the prevalence of Echinococcus spp. in dogs. METHODOLOGY/PRINCIPAL FINDINGS: PubMed, ScienceDirect, Chongqing VIP, China National Knowledge Infrastructure (CNKI), and WanFang databases were searched for relevant articles published in the past 10 years. A final total of 108 studies were included. The overall prevalence of Echinococcus spp. in dogs in China was 7.3%, with the highest point estimate found in sampling year 2015 (8.2%) and publication year 2015 (16.5%). Northwestern China (7.9%) had the highest infection rate in China. Qinghai Province (13.5%) showed the highest prevalence among the 11 provinces we included. We also found that geographical and climatic factors are related to the incidence of canine echinococcosis. We further investigated the source of heterogeneity by analysis of subgroups (sampling district, detection method, dog type, season, parasite species, medication, and study quality level). CONCLUSIONS/SIGNIFICANCE: Our research indicated that Echinococcus spp. were still prevalent in some areas in China. More localized prevention and control policies should be formulated, including improving drinking water hygiene and strengthening hygiene promotion. We recommend the rational use of anti-Echinococcus drugs. In addition, treatment of livestock offal and feces and improving the welfare of stray dogs may play an important role in reducing canine Echinococcus infections.

Lysine crotonylation is widespread on proteins of diverse functions and localizations in Toxoplasma gondii.

Lysine crotonylation (Kcr) is an evolutionally conserved post-translational modification (PTM) on histone proteins. However, information about Kcr and its involvement in the biology and metabolism of Toxoplasma gondii is limited. In the present study, a global Kcr proteome analysis using LC-MS/MS in combination with immune-affinity method was performed. A total of 12,152 Kcr sites distributed over 2719 crotonylated proteins were identified. Consistent with lysine acetylation and succinylation in Apicomplexa, Kcr was associated with various metabolic pathways, including carbon metabolism, pyrimidine metabolism, glycolysis, gluconeogenesis, and proteasome. Markedly, many stage-specific proteins, histones, and histone-modifying enzymes related to the stage transition were found to have Kcr sites, suggesting a potential involvement of Kcr in the parasite stage transformation. Most components of the apical secretory organelles were identified as crotonylated proteins which were associated with the attachment, invasion, and replication of T. gondii. These results expanded our understanding of Kcr proteome and proposed new hypotheses for further research of the Kcr roles in the pathobiology of T. gondii infection.

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.

Prevalence of porcine circovirus 2 throughout China in 2015-2019: A systematic review and meta-analysis.

Porcine circovirus type 2 (PCV2) causes infectious diseases in pigs leading to considerable economic losses in the pig industry. To prevent and control PCV2 infections, is important to understand the prevalence and geographical distribution of the virus. We performed the first systematic review and meta-analysis to estimate the prevalence of PCV2 in China. From PubMed, ScienceDirect, Chinese Web of Knowledge, Wanfang, and VIP Chinese Journal, we extracted 53 studies published in China between 2015 and 2019. There were 29,051 samples, 14,230 of which were positive for PCV2. The pooled prevalence of PCV2 was 46.0%, with the highest in Northeastern China (58.1%). The highest prevalence was 86.3% in Xinjiang province. Nursery pigs had the highest prevalence of PCV2 (50.9%), and the serological test detected the highest number of cases (58.5%). PCV2 prevalence was 50.1% in intensive farms and 37.5% in extensive farms. Our findings showed that PCV2 is common throughout China. Effective control measures are necessary to reduce PCV2 infections.

Prevalence and genotype distribution of Enterocytozoon bieneusi in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, eastern China.

Enterocytozoon bieneusi is a common microsporidian species, which can infect humans and various species of animals. However, little is known about E. bieneusi prevalence and genotypes in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, China. In this study, a total of 356 fecal samples were collected from farmed raccoon dogs in Weihai, Weifang, and Yantai cities in Shandong Province, China. A total of 23 (6.5%) samples were E. bieneusi-positive by nested PCR amplification of the internal transcribed spacer (ITS) region of ribosomal DNA. Statistical analysis showed that E. bieneusi prevalence in male raccoon dogs was higher than that in female raccoon dogs, and the highest E. bieneusi prevalence was detected in adult raccoon dogs. Sequence analysis revealed four known E. bieneusi genotypes (D, type IV, CHG1, and Peru8), and type IV (11/23) was the predominant genotype. The genotypes type IV, Peru8, and CHG1 were reported in raccoon dogs for the first time in China. Phylogenetic analysis showed that three human-pathogenic genotypes (D, type IV, and Peru8) were clustered into group 1, and the CHG1 belonged to group 2. These findings expand the current understanding of E. bieneusi prevalence and genotype distribution in raccoon dogs in China. Our study also shows that raccoon dogs are hosts for E. bieneusi belonging to several genotypes, including zoonotic ones, highlighting the possibility of transmission of this pathogen between raccoon dogs and humans.

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.

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.

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.

Molecular detection and genotype distribution of Enterocytozoon bieneusi in farmed silver foxes (Vulpes vulpes) and arctic foxes (Vulpes lagopus) in Shandong Province, eastern China.

Enterocytozoon bieneusi is an opportunistic enteric pathogen which can infect a wide range of animal species and humans. It is the most diagnosed species of Microsporidia in humans and has an impact on public health. Many infected animals including foxes may be a potential source for transmitting E. bieneusi to humans. However, limited information is available on the E. bieneusi prevalence and genotypes in farmed foxes in China. Therefore, in the present study, 344 fresh fecal samples were collected from farmed foxes (Vulpes vulpes and Vulpes lagopus) in Shandong Province, and the prevalence and genotypes of E. bieneusi were examined based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. The overall E. bieneusi prevalence was 9% (31/344); of them, 6.5% (9/138) in farmed silver foxes (V. vulpes) and 10.7% (22/206) in farmed arctic foxes (V. lagopus). Moreover, four known (Hum-q1, NCF2, HND-1, and Type IV) and two novel E. bieneusi genotypes (SDF1 and SDF2) were identified in farmed foxes in the present study. All of the E. bieneusi genotypes belonged to the zoonotic group based on phylogenetic analysis. In addition, 2, 4, 0, and 11 samples were successfully amplified at MS1, MS3, MS4, and MS7 loci, respectively. The present study reveals E. bieneusi prevalence and genotype distribution in farmed foxes in Shandong Province and enlarged the host and geographic information of E. bieneusi in China.

Serological evidence of hepatitis E virus and influenza A virus infection in farmed wild boars in China.

Hepatitis E virus (HEV) and influenza A virus (IAV) are two important pathogens which can infect humans and various animals causing public health problems. In this study, the seroprevalence and risk factors associated with HEV and IAV infection in farmed wild boars were investigated in China. A total of 758 serum samples were collected from farmed wild boars between 2015 and 2016, and antibodies against HEV and IAV were examined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits. The overall prevalence of anti-HEV antibodies was 24.54% (186/758, 95% CI 21.48-27.60) in farmed wild boars. There were statistically significant differences in the HEV seroprevalence in farmed wild boars of different ages (<22 days: 8.33%; 22-66 days: 18.89%; >66 days: 26.36%) (P < 0.05) and different genders (50.00% in male and 23.49% in female) (P < 0.01). However, there was no statistically significant difference in the HEV seroprevalence in farmed wild boars of different regions and different years. The overall IAV seroprevalence was 5.80% (44/758, 95% CI 4.14-7.46), and there was no statistically significant difference in the IAV seroprevalence in farmed wild boars of different ages and genders, collected from different regions and different years. Our results indicate that HEV and IAV infections in farmed wild boars may pose a potential risk for human infection. To our knowledge, this is the first report of HEV and IAV seroprevalence in farmed wild boars in China, which provides baseline data for further studies and for control of HEV and IAV infection in farmed wild boars.

First report of Neospora caninum seroprevalence in farmed raccoon dogs in China.

Neospora caninum is an intracellular protozoan parasite with a worldwide distribution, which can cause abortion and stillbirth in ruminants. However, little is known of N. caninum infection in raccoon dogs in China. Thus, a total of 1181 serum samples of farmed raccoon dogs were collected from the major production areas of Jilin, Changchun in Jilin province, and Rizhao, Weihai and Yantai in Shandong province in China. The antibodies to N. caninum were examined by a competitive-inhibition enzyme-linked immunoassay (cELISA). While the N. caninum seroprevalence in farmed raccoon dogs from different regions (cities) were not statistically significant, farmed raccoon dogs in Shandong province had a significantly higher N. caninum seroprevalence (8.99%, 95% CI 7.13-10.85) than those in Jilin province (4.46%, 95% CI 1.99-6.93) (P < 0.05). N. caninum seroprevalence in male and female raccoon dogs were 7.91% (95% CI 5.99-9.83) and 8.06% (95% CI 5.46-10.66), respectively. In addition, the seroprevalence of N. caninum infection in infancy, youth and adult raccoon dogs was 7.32% (95% CI 4.31-10.33), 7.93% (95% CI 5.44-10.42) and 8.41% (95% CI 5.82-11.00), respectively. This is the first report of N. caninum seroprevalence in farmed raccoon dogs in China, which provided baseline data for the prevention of N. caninum infection in raccoon dogs in China.

Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System.

Infection with the apicomplexan protozoan parasite Toxoplasma gondii is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of T. gondii pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 GRA genes (GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38, and GRA40) in the virulent T. gondii RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 GRA genes. Functional characterization of single ΔGRA mutants was achieved in vitro using cell-based plaque assay and egress assay, and in vivo in BALB/c mice. Targeted deletion of these 17 GRA genes had no significant effect neither on the in vitro growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17 GRA genes suggest that GRAs may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth in vitro or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations.

First Report of Seroprevalence and Risk Factors of Neospora caninum Infection in Tibetan Sheep in China.

Neospora caninum is an intracellular protozoan parasite which can cause abortion and stillbirth in ruminants. However, there is no information on Tibetan sheep N. caninum infection in China. A total of 2187 serum samples were collected from Tibetan sheep in the major production areas of Luqu, Maqu, and Tianzhu in Gansu province, and Nyingchi in southeast Tibet, China. All samples were analyzed for the presence of antibodies to N. caninum using a competitive-inhibition enzyme-linked immunoassay. Of the 2187 serum samples, 184 (8.4%, 95% CI 7.3-9.6) were tested N. caninum seropositive. The N. caninum seroprevalence ranged from 4.4% (95% CI 1.4-7.4) to 11.3% (95% CI 8.2-14.4) among different regions, seasons, ages, and pregnancies, and there was no statistical significance among those groups (P > 0.05). Seroprevalence in male (10.8% 69/638) (95% CI 8.4-13.2) was significantly higher than in female (7.4% 115/1549) (OR =1.51, 95% CI 6.1-8.7) (P < 0.01). To our knowledge, this is the first report of N. caninum seroprevalence in Tibetan sheep in China, which provides baseline data for the prevention and control of N. caninum infection in Tibetan sheep.

First Report of Chlamydia Seroprevalence in Farmed Wild Boars in China.

Chlamydia is Gram-negative obligate bacterium, which can cause human diseases worldwide and has huge economic impact on animals. It is yet to know whether farmed wild boars are infected with Chlamydia in China. To assess risk factors of Chlamydia infection in farmed wild boars in China, from April 2015 to February 2016, a total of 837 serum samples of farmed wild boars were collected in Jilin province, northeastern China, and antibodies against Chlamydia were examined by the indirect hemagglutination assay. The investigation showed that antibodies to Chlamydia were detected in 332 (39.67%, 95% CI 33.36-42.98) of 837 serum samples of farmed wild boars, seroprevalence ranged from 33.71% to 44.42% among different regions and the differences were statistically significant by SPSS analysis (p = 0.0248). These results indicated that Chlamydia is highly prevalent in farmed wild boars in Jilin province, northeastern China, and may pose a potential risk for human health. To our knowledge, this is the first report of Chlamydia seroprevalence in farmed wild boars in China, which provided baseline data for preventing and controlling Chlamydia infection in wild boars in China.

First report of Mycoplasma hyopneumoniae seroprevalence in farmed wild boars in China.

Porcine enzootic pneumonia caused by Mycoplasma hyopneumoniae affects the global pig industry with significant economic losses. It is yet to know whether wild boars in China were infected with M. hyopneumoniae. The present study was conducted to examine the seroprevalence and to evaluate risk factors of M. hyopneumoniae infection in farmed wild boars in China. A total of 882 serum samples were collected from farmed wild boars in Jilin City, Siping City and Baishan City in Jilin Province, northeastern China from April 2015 to February 2016, and were examined by the double sandwich enzyme-linked immunosorbent assay (ELISA). Seventy-eight out of 882 (8.8%) serum samples were M. hyopneumoniae-seropositive. Among region groups, wild boars from Jilin city (11.7%, 33/281) had the highest seropositivity, followed by Siping city (11%, 29/263) and Baishan city (4.7%, 16/338), and the difference was statistically significant (P = 0.0031). The M. hyopneumoniae seroprevalence in the female wild boars (9.0%, 75/831) was higher than that in the male wild boars (5.9%, 3/51) (P = 0.4429). The results of this investigation showed that farmed wild boars were susceptible to M. hyopneumoniae. Logistic regression analysis showed that there is a significant correlation between the geographical area and M. hyopneumoniae infection, which may be related to the regional environment. This is the first report of M. hyopneumoniae seroprevalence in farmed wild boars in China, which provided baseline information for further studies and control of M. hyopneumoniae infection in wild boars in China.