Update on the genetic diversity and population structure of Echinococcus granulosus in Gansu Province, Tibet Autonomous Region, and Xinjiang Uygur Autonomous Region, Western China, inferred from mitochondrial cox1, nad1, and nad5 sequences.

The identification of additional Echinococcus granulosus sensu lato (s.l.) complex species/genotypes in recent years raises the possibility that there might be more variation among this species in China than is currently understood. The aim of this study was to explore intra- and inter-species variation and population structure of Echinococcus species isolated from sheep in three areas of Western China. Of the isolates, 317, 322, and 326 were successfully amplified and sequenced for cox1, nad1, and nad5 genes, respectively. BLAST analysis revealed that the majority of the isolates were E. granulosus s.s., and using the cox1, nad1, and nad5 genes, respectively, 17, 14, and 11 isolates corresponded to Elodea canadensis (genotype G6/G7). In the three study areas, G1 genotypes were the most prevalent. There were 233 mutation sites along with 129 parsimony informative sites. A transition/transversion ratio of 7.5, 8, and 3.25, respectively, for cox1, nad1, and nad5 genes was obtained. Every mitochondrial gene had intraspecific variations, which were represented in a star-like network with a major haplotype with observable mutations from other distant and minor haplotypes. The Tajima's D value was significantly negative in all populations, indicating a substantial divergence from neutrality and supporting the demographic expansion of E. granulosus s.s. in the study areas. The phylogeny inferred by the maximum likelihood (ML) method using nucleotide sequences of cox1-nad1-nad5 further confirmed their identity. The nodes assigned to the G1, G3, and G6 clades as well as the reference sequences utilized had maximal posterior probability values (1.00). In conclusion, our study confirms the existence of a significant major haplotype of E. granulosus s.s. where G1 is the predominant genotype causing of CE in both livestock and humans in China.

A new molecular nomenclature for Taenia hydatigena: mitochondrial DNA sequences reveal sufficient diversity suggesting the assignment of major haplotype divisions.

Cysticercosis caused by the metacestode larval stage of Taenia hydatigena formerly referred to as Cysticercus tenuicollis is a disease of veterinary importance that constitutes a significant threat to livestock production worldwide, especially in endemic regions due to condemnation of visceral organs and mortality rate of infected young animals. While the genetic diversity among parasites is found to be potentially useful in many areas of research including molecular diagnostics, epidemiology and control, that of T. hydatigena across the globe remains poorly understood. In this study, analysis of the mitochondrial DNA (mtDNA) of adult worms and larval stages of T. hydatigena isolated from dogs, sheep and a wild boar in China showed that the population structure consists of two major haplogroups with very high nucleotide substitutions involving synonymous and non-synonymous changes. Compared with other cestodes such as Echinococcus spp., the genetic variation observed between the haplogroups is sufficient for the assignment of major haplotype or genotype division as both groups showed a total of 166 point-mutation differences between the 12 mitochondrial protein-coding gene sequences. Preliminary analysis of a nuclear protein-coding gene (pepck) did not reveal any peculiar changes between both groups which suggests that these variants may only differ in their mitochondrial makeup.

Molecular detection of Blastocystis in black bears and sika deer in northern China.

Protists of the Blastocystis genus are distributed worldwide and can infect a range of hosts. However, data concerning Blastocystis infection are limited for sika deer and are not available for black bears. Therefore, in the present study, a total of 312 black bears (Ursus thibetanus) from Heilongjiang Province and 760 sika deer (Cervus nippon) from four different northern Chinese provinces were investigated. Blastocystis infection in these animals was detected via PCR amplification of the small subunit rRNA gene in fecal samples. The prevalence of Blastocystis infection in black bears and sika deer was 14.4% (45/312 positive samples) and 0.8% (6/760 positive samples), respectively. Young black bears (18.3%) had a significantly higher Blastocystis prevalence than adult bears (9.1%). The prevalence of Blastocystis was significantly higher in black bears raised outdoors (24.6%) than in bears raised indoors (12.2%). Blastocystis-positive sika deer were only found in Jilin Province (1.3%, 6/480). Female sika deer (0%, 0/61) had a significantly lower Blastocystis prevalence than males (0.9%, 6/699). Sanger sequencing was used to determine the small subunit rRNA gene sequences of the Blastocystis-positive PCR products. A neighbor-joining phylogenetic tree based on the small subunit rRNA gene sequences showed that only Blastocystis subtype (ST)1 was identified in black bears, whereas ST10 and ST14 were found in sika deer. This is the first report of Blastocystis ST1 infection in black bears. These findings also extend the distribution information of Blastocystis subtypes, which will provide a foundation for further study of Blastocystis in different hosts in China.

Prevalence, risk factors and genotype distribution of Toxoplasma gondii DNA in soil in China.

In the present study, we performed a cross-sectional survey to determine the occurrence and genotype distribution of T. gondii DNA in soil samples collected from different sources from six geographic regions in China. Between March 2015 and June 2017, 2100 soil samples were collected from schools, parks, farms and coastal beaches, and examined for T. gondii DNA using three PCR assays targeting 529-bp repeat element (RE) sequence, B1 gene and ITS-1 gene sequences. Also, we investigated whether geographic region, soil source and type, and sampling season can influence the prevalence of T. gondii DNA in the soil. Soil samples collected from farms and parks had the highest prevalence, whereas samples collected from school playgrounds and coastal beaches had the lowest prevalence. PCR assays targeting 529-bp RE and ITS-1 gene sequences were more sensitive than the B1 gene-based assay. Positive PCR products were genotyped using multi-locus PCR-RFLP, and ToxoDB #9 was the predominant genotype found in the contaminated soil samples. Multiple logistic regression identified factors correlated significantly with the presence of T. gondii DNA in the soil to be the source of the soil, including farms (odds ratio 3.10; 95% confidence interval [CI], 1.52 to 6.29; p = 0.002) and parks (2.59; 95% CI 1.28 to 5.27; p = 0.009). These results show that Chinese soil hosts T. gondii of the most prevalent genotype in China (ToxoDB#9) and that the soil type influences infection patterns.

Detection and genetic characterization of Toxoplasma gondii in market-sold mussels (Mytilus edulis) in certain provinces of China.

Mussels, randomly collected from fish markets in China, were analyzed by a semi-nested PCR to detect B1 gene of Toxoplasma gondii. Out of the 2215 samples, fifty-five (2.48%) were detected T. gondii-positive. The prevalence in Shandong province, Liaoning province and Zhejiang province were 2.51%, 2.26% and 2.69%, respectively. T. gondii oocysts were more frequently detected in digestive glands (1.04%) and haemolymph (1.49%) when compared with gills (0.23%). Of the 55 positive DNA, only two samples showed complete genotype at 11 locus and were authenticated as ToxoDB Genotype #9. To our knowledge, the present study is the first to confirm the presence of T. gondii in market-sold mussels in China. The findings point to the risk of humans acquiring T. gondii infection by consuming mussels bought in the aquatic product market.

Prevalence of Cryptosporidium in dairy cattle in China during 2008-2018: A systematic review and meta-analysis.

Cryptosporidium is the causative agent of cryptosporidiosis. Cryptosporidium not only has a worldwide distribution, but also can infect various hosts, including dairy cattle and humans. Although numerous researches on Cryptosporidium infection in cattle have been conducted, no nationwide study on the prevalence of Cryptosporidium infection in dairy cattle in mainland China was carried out. In this meta-analysis, five databases, including PubMed, ScienceDirect, China National Knowledge Infrastructure (CNKI), Chongqing VIP, and Wanfang, were used to search for published papers regarding Cryptosporidium infection in dairy cattle in China from inception to February 25, 2019. Our study obtained 60 eligibility studies that reported Cryptosporidium infection in dairy cattle. We estimated the pooled Cryptosporidium prevalence to be 17.0% (3,901/33,313), with 16.9% (722/5,191) in Central China, 17.4% (959/6,162) in Eastern China, 29.8% (404/2,021) in Northeastern China, 15.7% (227/2,344) in Northern China, 15.8% (1,042/11,452) in Northwestern China, 9.5% (494/5,758) in Southern China, and 13.7% (53/385) in Southwestern China. The pooled prevalence of Cryptosporidium in before 2000 group (28.0%, 944/3,417) was significantly higher than in 2000-2010 group (11.1%, 384/3,643) and after 2010 group (13.7%, 2,134/22,411). Cattle with the age of ≤ 12 months (22.5%, 2,142/12,948) had a significantly higher prevalence than those of > 12 months (9.5%, 840/10,282). The pooled prevalence of Cryptosporidium in different seasons ranged from 8.2% (343/4,552) in Autumn to 19.5% (285/1,570) in Winter. Diarrhea cattle (38%, 133/477) had a higher Cryptosporidium prevalence than non-diarrhea cattle (13.0%, 367/2423). The pooled prevalence of Cryptosporidium in different provinces was various, with the highest (35.6%, 355/1,667) in Heilongjiang province, and the lowest (4.3%, 15/440) in Tianjin province. The univariate meta-regression analyses indicated that the collection year (P = 0.002) and age of cattle (P = 0.001) might be sources of heterogeneity. This systematic review suggests that China is a country where cryptosporidiosis frequently occurs in cattle. Due to the particular relationship between dairy cattle and feeder, further research is required to investigate the links between cattle ownership and Cryptosporidium infection.

A multiplex PCR assay for the simultaneous detection of Taenia hydatigena, T. multiceps, T. pisiformis, and Dipylidium caninum infections.

BACKGROUND: Taenia hydatigena, T. multiceps, T. pisiformis, and Dipylidium caninum are four common large and medium-sized tapeworms parasitizing the small intestine of dogs and other canids. These parasites cause serious impact on the health and development of livestock. However, there are, so far, no commercially available molecular diagnostic kits capable of simultaneously detecting all four parasites in dogs. The aim of the study was therefore to develop a multiplex PCR assay that will accurately detect all four cestode infections in one reaction. METHODS: Specific primers for a multiplex PCR were designed based on corresponding mitochondrial genome sequences, and its detection limit was assessed by serial dilutions of the genomic DNAs of tapeworms examined. Furthermore, field samples of dog feces were tested using the developed assay. RESULTS: A multiplex polymerase chain reaction (PCR) assay was developed based on mitochondrial DNA (mtDNA) that accurately and simultaneously identify four cestode species in one reaction using specific fragment sizes of 592, 385, 283, and 190 bp for T. hydatigena, T. multiceps, T. pisiformis, and D. caninum, respectively. The lowest DNA concentration detected was 1 ng for T. hydatigena, T. multiceps and T. pisiformis, and 0.1 ng for D. caninum in a 25 µl reaction system. This assay offers high potential for the rapid detection of these four tapeworms in host feces simultaneously. CONCLUSIONS: This study provides an efficient tool for the simultaneous detection of T. hydatigena, T. multiceps, T. pisiformis, and D. caninum. The assay will be potentially useful in epidemiological studies, diagnosis, and treatment of these four cestodes infections during prevention and control program.

Immune response and protective effect against chronic Toxoplasma gondii infection induced by vaccination with a DNA vaccine encoding profilin.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals. T. gondii profilin (TgPF) plays a crucial role in parasite motility and host cell invasion, and has shown promise against toxoplasmosis. DNA vaccine was considered to elicit effective humoral and cell-mediated immunity against T. gondii infection. The objective of the present study was to evaluate the immunogenicity of TgPF in mice using a DNA vaccination strategy. METHODS: A DNA vaccine (pVAX-PF) encoding TgPF gene was constructed and then was intramuscularly injected into mice with and without a plasmid encoding IL-15 (pVAX-IL-15). The immune responses in immunized Kunming mice including lymphocyte proliferation, levels of cytokines, antibody titers and T lymphocyte subclasses were analyzed. The protective efficacy against chronic T. gondii infection was observed at 4 weeks post-infection with the cyst-forming PRU strain of T. gondii (Genotype II). RESULTS: EitherpVAX-PF with or without pVAX-IL-15 could elicit higher level of IgG and IgG2a antibodies and produce strong cellular immune responses in the immunized mice. The brain cyst numbers in mice immunized with pVAX-PF + pVAX-IL-15 (1843 ± 215.7) and pVAX-PF (1897 ± 337.8) were reduced 40.82% and 39.08%, respectively, compared to that in mice received nothing (3114 ± 168.8), and the differences were statistically significant (P < 0.0001). However, the T. gondii cyst numbers in mice immunized with pVAX-PF + pVAX-IL-15 were not statistically significantly different compared to that in mice immunized with pVAX-PF alone [t(10) = 0.33, P > 0.05]. CONCLUSIONS: The present study indicated that TgPF could be a promising vaccine candidate against chronic toxoplasmosis, which can be further used to develop multi-epitope vaccine formulations in food-producing animals against T. gondii infection.

Immune Responses Induced by HSP60 DNA Vaccine against Toxoplasma gondii Infection in Kunming Mice.

Toxoplasma gondii can infect all the vertebrates including human, and leads to serious toxoplasmosis and considerable veterinary problems. T. gondii heat shock protein 60 (HSP60) is associated with the activation of antigen presenting cells by inducing initial immune responses and releasing inflammatory cytokines. It might be a potential DNA vaccine candidate for this parasite. A pVAX-HSP60 DNA vaccine was constructed and immune responses was evaluated in Kunming mice in this study. Our data indicated that the innate and adaptive immune responses was elicited by successive immunizations with pVAX-HSP60 DNA, showing apparent increases of CD3e+CD4+ and CD3e+CD8a+ T cells in spleen tissues of the HSP60 DNA-immunized mice (24.70±1.23% and 10.90±0.89%, P<0.05) and higher levels of specific antibodies in sera. Furthermore, the survival period of the immunized mice (10.53±4.78 day) were significantly prolonged during the acute T. gondii infection. Decrease of brain cysts was significant in the experimental group during the chronic infection (P<0.01). Taken together, TgHSP60 DNA can be as a vaccine candidate to prevent the acute and chronic T. gondii infections.

Vaccination with Toxoplasma gondii calcium-dependent protein kinase 6 and rhoptry protein 18 encapsulated in poly(lactide-co-glycolide) microspheres induces long-term protective immunity in mice.

BACKGROUND: Toxoplasmosis is a worldwide zoonosis caused by the intracellular parasite Toxoplasma gondii. However, no effective vaccine is yet available. Poly(lactide-co-glycolide) polymers can reduce protein degradation and sustain the release of antigens over a long period, which could generate a long-lasting immune response in vivo. Using a mouse model of toxoplasmosis, we evaluated the protective efficacy of vaccination with two recombinant proteins, which are formulated in biodegradable polymers. METHODS: Two recombinant proteins, rCDPK6 and rROP18, were encapsulated in poly(D,L-lactide-co-glycolide) (PLG), and then injected subcutaneously into Kunming mice. The mice immune responses were evaluated in terms of lympho-proliferation, cytokine expression, and antibodies. The survival of infected mice and brain cyst formation were also evaluated at 6 weeks after challenge with T. gondii RH strain (genotype I) or PRU strain (genotype II). RESULTS: Both protein vaccines induced Th1-biased immune responses, with increased specific antibodies and T cells, high levels of interferon-γ and interleukin 2, and strong lymphocyte proliferative responses. The mice immunized with the various protein vaccines survived slightly longer time than the control groups (P > 0.05) after injection with T. gondii RH strain. There were fewer brain cysts in the mice in all the immunized groups than that in the control groups, and the brain cysts were significantly reduced in mice immunized with proteins + 206, rCDPK6 + PLG and rCDPK6 + rROP18 + PLG (P < 0.05) compared controls. Further comparison of the immune responses to the proteins adjuvanted with PLG or Montanide™ ISA 206 VG 6 weeks after the last immunization revealed that antigens encapsulated in PLG conferred greater protective immunity against challenge. CONCLUSIONS: These findings suggest that the two recombinant T. gondii proteins encapsulated in PLG conferred immunity to T. gondii for an extended period, providing the foundation for the further development of a commercial vaccine against toxoplasmosis.

A long-lasting protective immunity against chronic toxoplasmosis in mice induced by recombinant rhoptry proteins encapsulated in poly (lactide-co-glycolide) microparticles.

Toxoplasma gondii infection in humans and animals is a worldwide zoonosis. Prevention and control of toxoplasmosis based on vaccination is one of the promising strategies. In the present study, recombinant T. gondii rhoptry proteins 38 and 18 (TgROP38 and TgROP18) were encapsulated into poly (lactide-co-glycolide) (PLG) (1:1), respectively, to obtain the stable water-in-oil-in-water double emulsion. Female Kunming mice were then immunized with the protein vaccines twice at a 2-week interval. Eight weeks after the second immunization, 10 mice from each group were challenged with T. gondii PRU strain (genotype II). The entrapment rates of PLG-rROP38 and PLG-rROP18 ranged from 65.5 to 77.7% and 58.1 to 72.3%, respectively. Immunization of mice with rROP38 and rROP18 proteins encapsulated into PLG microparticles elicited strongly humoral and cell-mediated responses against T. gondii, associated with relatively high levels of total IgG, IgG2a isotype, and IFN-γ, as well as the mixed Th1/Th2 immunity responses. Immunization with various protein vaccines induced significant reduction of the brain cysts after chronic infection with the T. gondii PRU strain, and the most effective protection was achieved in the PLG-rROP38-rROP18-immunized mice, with a cyst reduction of 81.3%. The findings of the present study indicated that recombinant rhoptry antigens encapsulated in PLG could maintain the protein immunogenicity in an extended period and elicit effective protection against chronic T. gondii infection, which has implications for the development of long-lasting vaccines against chronic toxoplasmosis in animals.

Evaluation of immuno-efficacy of a novel DNA vaccine encoding Toxoplasma gondii rhoptry protein 38 (TgROP38) against chronic toxoplasmosis in a murine model.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite which can infect almost all mammalian animals, leading to toxoplasmosis. T. gondii rhoptry protein 38 (TgROP38) is an active rhoptry protein kinase which is involved in the inhibitory effect on host cell transcription by down-regulating the MAPK signaling track. METHODS: TgROP38 gene was amplified and inserted into eukaryotic vector pVAX I and formed the DNA vaccine pVAX-ROP38. Mice in the experimental group were intramuscularly immunized with pVAX-ROP38 and those injected with pVAX I, PBS or nothing were treated as controls. After three injections at two week intervals, all mouse groups were challenged intraperitoneally with 1000 tachyzoites of the virulent T. gondii RH strain (Type I, ToxoDB #10) and 10 cysts of the PRU strain (Type II, ToxoDB #1), respectively. RESULTS: Mice inoculated with pVAX-ROP38 vaccine had a higher level of IgG antibodies (P < 0.01) and T lymphoproliferative response. The high ratio of IgG2a/IgG1 and the increasing levels of IFN-γ and IL-2 (P < 0.05) indicated an activated Th1 cell-mediated immune responses. Furthermore, the CD4+ and CD8+ proportions in vaccinated mice were also increased significantly compared with that in mice of the three control groups (P < 0.01). In the model of acute infection, the average survival time of mice in the pVAX-ROP38 group (8.1 days ± 0.75) was no statistically different compared to that in the PBS, pVAX I and blank control groups which died within 7 days. However, in the model of chronic infection, the brain cyst reduction in the pVAX-ROP38 group reached 76.6%, compared to controls (P < 0.01). CONCLUSIONS: The present study revealed that the pVAX-ROP38 vaccine could elicit strong humoral and cell immunity response against chronic T. gondii infection in mice, resulting in the reduction of the brain cyst formation effectively, which suggests that TgROP38 is a desirable vaccine candidate against chronic T. gondii infection.

Detection of Toxoplasma gondii oocysts in soils in northwestern China using a new semi-nested PCR assay.

BACKGROUND: Toxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China. RESULTS: The one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 10(2) T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0-1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%. CONCLUSIONS: The present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.

Sequence variation in Toxoplasma gondii rop17 gene among strains from different hosts and geographical locations.

Genetic diversity of T. gondii is a concern of many studies, due to the biological and epidemiological diversity of this parasite. The present study examined sequence variation in rhoptry protein 17 (ROP17) gene among T. gondii isolates from different hosts and geographical regions. The rop17 gene was amplified and sequenced from 10 T. gondii strains, and phylogenetic relationship among these T. gondii strains was reconstructed using maximum parsimony (MP), neighbor-joining (NJ), and maximum likelihood (ML) analyses. The partial rop17 gene sequences were 1375 bp in length and A+T contents varied from 49.45% to 50.11% among all examined T. gondii strains. Sequence analysis identified 33 variable nucleotide positions (2.1%), 16 of which were identified as transitions. Phylogeny reconstruction based on rop17 gene data revealed two major clusters which could readily distinguish Type I and Type II strains. Analyses of sequence variations in nucleotides and amino acids among these strains revealed high ratio of nonsynonymous to synonymous polymorphisms (>1), indicating that rop17 shows signs of positive selection. This study demonstrated the existence of slightly high sequence variability in the rop17 gene sequences among T. gondii strains from different hosts and geographical regions, suggesting that rop17 gene may represent a new genetic marker for population genetic studies of T. gondii isolates.

The transcription factor AP2XI-2 is a key negative regulator of Toxoplasma gondii merogony.

Sexual development in Toxoplasma gondii is a multistep process that culminates in the production of oocysts, constituting approximately 50% of human infections. However, the molecular mechanisms governing sexual commitment in this parasite remain poorly understood. Here, we demonstrate that the transcription factors AP2XI-2 and AP2XII-1 act as negative regulators, suppressing merozoite-primed pre-sexual commitment during asexual development. Depletion of AP2XI-2 in type II Pru strain induces merogony and production of mature merozoites in an alkaline medium but not in a neutral medium. In contrast, AP2XII-1-depleted Pru strain undergoes several rounds of merogony and produces merozoites in a neutral medium, with more pronounced effects observed under alkaline conditions. Additionally, we identified two additional AP2XI-2-interacting proteins involved in repressing merozoite programming. These findings underscore the intricate regulation of pre-sexual commitment by a network of factors and suggest that AP2XI-2 or AP2XII-1-depleted Pru parasites can serve as a model for studying merogony in vitro.

Protective immunity against Toxoplasma gondii induced by DNA immunization with the gene encoding a novel vaccine candidate: calcium-dependent protein kinase 3.

BACKGROUND: Toxoplasma gondii can infect almost all warm-blood animals including human beings. The plant-like calcium-dependent protein kinases (CDPKs) harbored by T. gondii are involved in gliding motility, cell invasion, egress and some other developmental processes, and so have been implicated as important virulence factors. METHODS: In the present study, we constructed a DNA vaccine expressing T. gondii CDPK3 (TgCDPK3) and evaluated its protective efficacy against T. gondii infection in Kunming mice. The gene sequence encoding TgCDPK3 was inserted into the eukaryotic expression vector pVAX I, and mice were immunized with pVAX-CDPK3 intramuscularly. RESULTS: The results showed that mice immunized with pVAX-CDPK3 developed a high level of specific antibodies and a strong lymphoproliferative response. The significantly increased levels of IFN-γ, IL-2, IL-12 (p70) and IL-23 and high ratio of IgG2a to IgG1 antibody titers indicated that a Th1 type response was elicited after immunization with pVAX-CDPK3. Furthermore, the percentage of CD4+ T cells in mice vaccinated with pVAX-CDPK3 was significantly increased. After lethal challenge with the tachyzoites of the virulent T. gondii RH strain, the mice immunized with pVAX-CDPK3 prolonged the survival time from 10 days to 24 days (13.5 ± 4.89) compared to untreated mice or those received PBS or pVAX I which died within 7 days (P < 0.05). In chronic infection model (10 cysts of the T. gondii PRU strain), the numbers of brain cysts of the mice immunized with pVAX-CDPK3 reduced significantly when compared with those in control groups (P < 0.05), and the rate of reduction could reach to about 50%. CONCLUSIONS: TgCDPK3 can generate protective immunity against acute and chronic T. gondii infection in Kunming mice and is a promising vaccine candidate for further development of an effective vaccine against T. gondii.

Prevalence and genetic characterization of Toxoplasma gondii in house sparrows (Passer domesticus) in Lanzhou, China.

The prevalence of Toxoplasma gondii infection in birds has epidemiological significance because birds are indeed considered as a good indicator of environmental contamination by T. gondii oocysts. In this study, the prevalence of T. gondii in 313 house sparrows in Lanzhou, northwestern China was assayed by the modified agglutination test (MAT). Antibodies to T. gondii were positive in 39 (12.46%) of 313 samples (MAT titer ≥ 1:5). Tissues of heart, brain, and lung from the 39 seropositive house sparrows were tested for T. gondii DNA, 11 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 9 genetic markers, including 8 nuclear loci, i.e., SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8 and an apicoplast locus Apico. Of them, 4 isolates were genotyped with complete data for all loci, and 2 genotypes (Type II variants; ToxoDB #3 and a new genotype) were identified. These results showed that there is a potential risk for human infection with T. gondii in this region. To our knowledge, this is the first report of T. gondii seroprevalence in house sparrows in China.

Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis.

BACKGROUND: In the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China. METHODS: From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed. RESULTS: The isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis. CONCLUSIONS: As a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.

Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro.

Toxoplasmosis, caused by Toxoplasma gondii, is a common disease worldwide and could be severe and even fatal in immunocompromised individuals and fetuses. Limitation in current available treatment options drives the need to develop novel therapeutics. This study assessed the anti-T. gondii potential of 103 marine natural products. A luminescence-based ß-galactosidase activity assay was used to screen the marine natural products library. Afterward, those compounds that displayed over 70% parasite inhibition ratio were further chosen to assess their cytotoxicity. Compounds exhibiting low cytotoxicity (≥80% cell viability) were applied to evaluate the inhibition efficacy on discrete steps of the T. gondii lytic cycle, including invasion, intracellular growth, and egress abilities as well as the cell cycle. We found that both estradiol benzoate and octyl gallate caused >70% inhibition of tachyzoite growth with IC50 values of 4.41 ± 0.94 and 5.66 ± 0.35 µM, respectively, and displayed low cytotoxicity with TD50 values of 34.11 ± 2.86 and 26.4 ± 0.98 µM, respectively. Despite their defects in inhibition of invasion and egress of tachyzoite, the two compounds markedly inhibited the tachyzoite intracellular replication. Flow cytometric analyses further suggested that the anti-T. gondii activity of estradiol benzoate, rather than octyl gallate, may be linked to halting cell cycle progression of tachyzoite from G1 to S phase. Taken together, these findings suggest that both estradiol benzoate and octyl gallate are potential inhibitors for anti-T. gondii infection and support the further exploration of marine natural products as a thinkable source of alternative and active agents against T. gondii.

Novel study on the prevalence of Trichinella spiralis in farmed American minks (Neovison vison) associated with exposure to wild rats (Rattus norvegicus) in China.

Minks and brown rats are reservoir hosts for many endoparasites including those of the genus Trichinella, a group of parasite nematodes with a worldwide distribution. However, little is known about the prevalence of Trichinella sp. infection in the American mink (Neovison vison) and rats (Rattus norvegicus) in China. Therefore, we aimed to examine the prevalence of Trichinella sp. infection in farmed minks in Weihai city, Shandong province, China and infer the possible route for Trichinella transmission to farmed American minks. In total, 289 muscle samples from minks and 102 carcasses of rats were collected from Weihai City. The appearance of Trichinella sp. was examined using the pooled artificial HCl-pepsin digestion method. The results showed that muscle larvae were detected in 20 of 289 minks (6.92%) and 2 of 102 synanthropic rats (1.96%). The larval density of Trichinella sp. in mink samples ranged from 0.025 to 0.815 larvae per gram (lpg), while the average larval burden in rats was 0.17 lpg. The isolates derived from minks and rats were identified at the species level using multiplex polymerase chain reaction (PCR), which revealed that the size of the two PCR products matched that of T. spiralis at 173 bp. Furthermore, sequence analysis showed 100% identity of the 5S rDNA inter-gene spacer regions of the two isolates to that of T. spiralis. This study presents a novel report of T. spiralis-mediated infection in minks and synanthropic rats in China. We highlight the vulnerability of farmed minks to Trichinella infection through exposure to synanthropic rats, which may raise a public health concern of potential zoonotic risks for domestic animals.