Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

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.

A newly characterized dense granule protein (GRA76) is important for the growth and virulence of Toxoplasma gondii.

Pathogenicity of the zoonotic pathogen Toxoplasma gondii largely depends on the secretion of effector proteins into the extracellular milieu and host cell cytosol, including the dense granule proteins (GRAs). The protein-encoding gene TGME49_299780 was previously identified as a contributor to parasite fitness. However, its involvement in parasite growth, virulence and infectivity in vitro and in vivo remains unknown. Here, we comprehensively examined the role of this new protein, termed GRA76, in parasite pathogenicity. Subcellular localization revealed high expression of GRA76 in tachyzoites inside the parasitophorous vacuole (PV). However, its expression was significantly decreased in bradyzoites. A CRISPR-Cas9 approach was used to knock out the gra76 gene in the T. gondii type I RH strain and type II Pru strain. The in vitro plaque assays and intracellular replication showed the involvement of GRA76 in replication of RH and Pru strains. Deletion of the gra76 gene significantly decreased parasite virulence, and reduced the brain cyst burden in mice. Using RNA sequencing, we detected a significant increase in the expression of bradyzoite-associated genes such as BAG1 and LDH2 in the PruΔgra76 strain compared with the wild-type Pru strain. Using an in vitro bradyzoite differentiation assay, we showed that loss of GRA76 significantly increased the propensity for parasites to form bradyzoites. Immunization with PruΔgra76 conferred partial protection against acute and chronic infection in mice. These findings show the important role of GRA76 in the pathogenesis of T. gondii and highlight the potential of PruΔgra76 as a candidate for a live-attenuated vaccine.

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.

A Strainer-Based Platform for the Collection and Immunolabeling of Porcine Epidemic Diarrhea Virus-Infected Porcine Intestinal Organoid.

The development of organoid research has raised new requirements for this methodology. In a previous study, we demonstrated that an emerging protocol achieved the collection, loading, and programmed immunolabeling of mouse intestinal organoids based on a strainer platform. To uncover the applied potential of this novel methodology on organoids from other species, the strainer platform was utilized to characterize the porcine epidemic diarrhea virus (PEDV)-infected porcine intestinal organoid model. Based on a previous study, some steps were changed to improve the efficiency of the assay by simplifying the reagent addition procedure. In addition, we redefined the range of strainer sizes on porcine intestinal organoids, showing that strainers with pore sizes of 40 and 70 µm matched the above protocol well. Notably, the strainer platform was successfully used to label viral proteins, laying the foundation for its application in the visualization of viral infection models. In summary, the potential of the strainer platform for organoid technology was explored further. More extensive exploration of this platform will contribute to the development of organoid technology.

A Strainer-Based Platform for the Collection and Immunolabeling of Mouse Intestinal Organoids.

Intestinal organoids have emerged as powerful model systems for studying the complex structure and function of the intestine. However, there is a lack of widely applicable methods for the collection, labeling, and imaging of intestinal organoids. In this study, we developed a novel method for loading and labeling intestinal organoids, a method that efficiently collects the organoids and facilitates imaging of their three-dimensional (3D) structure. Based on this strainer platform, mouse intestinal organoids were adequately collected and immobilized, facilitating the immunolabeling workflow to target proteins of the organoids. After evaluation, the strainer size of 40 µm was considered to be more conducive to the collection and labeling of mouse intestinal organoids. More extensive research on organoids of multiple types and species origins will contribute to broadening the applicability of the methodology. Overall, our study proposes an innovative workflow for loading and analyzing intestinal organoids. The combination of a strainer-based collection method, fluorescent labeling, and 3D reconstruction provides valuable insights into the organization and complexity of these tissue models, thereby offering new avenues for investigating intestinal development, disease modeling, and drug discovery.

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.

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, ß-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). ß-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Genetic variation and population structure of Fasciola hepatica: an in silico analysis.

Fasciola hepatica is a trematode leading to heavy economic setbacks to the livestock sector globally. The population's genetic information and intimate kinship level are frequently assessed using analysis of mitochondrial DNA. In this analysis, we retrieved cox1 (n = 247) and nad1 (n = 357) sequences of F. hepatica from the NCBI GenBank database and aligned the sequences with the respective reference sequences using MEGA software. The median joining network was drawn using PopArt software while neutrality and diversity indices were estimated with the help of DnaSp software. Neighbor-joining phylogenetic tree was constructed using the MEGA software package. A total of 46 and 98 distinctive haplotypes were observed for cox1 and nad1 genes, respectively. Diversity indices indicated high haplotype and nucleotide diversities in both genes. Positive Tajima's D and Fu's Fs values were found for the entire population of both the genes under study. The cox1 and nad1 gene segments in this study showed high Tajima's D values, suggesting a low likelihood of future population growth. The Tajima's D value of the nad1 gene sequence is lower (2.14910) than that of the cox1 gene sequence (3.40314), which suggests that the former is growing at a slower rate. However, the region-wise analysis revealed that both the cox1 and nad1 genes showed deviation from neutrality suggesting a recent population expansion as a result of an excess of low-frequency polymorphism. Furthermore, the overall host-wise analysis showed positive and significant Tajima's D values for the cox1 and nad1 gene sequences. To the best of our knowledge, this is the first attempt to provide insights into genetic variations and population structure of F. hepatica at a global scale using cox1 and nad1 genes. Our findings suggest the existence of specific variants of F. hepatica in different parts of the world and provide information on the molecular ecology of F. hepatica. The results of this study also mark a critical development in upcoming epidemiological investigations on F. hepatica and will also contribute to understanding the global molecular epidemiology and population structure of F. hepatica.

Establishment of a secondary infection laboratory model of Echinococcus shiquicus metacestode using BALB/c mice and Mongolian jirds (Meriones unguiculatus).

Echinococcus shiquicus is peculiar to the Qinghai­Tibet plateau of China. Research on this parasite has mainly focused on epidemiological surveys and life cycle studies. So far, limited laboratory studies have been reported. Here, experimental infection of E. shiquicus metacestode in BALB/c mice and Mongolian jirds (Meriones unguiculatus) was carried out to establish alternative laboratory animal models. Intraperitoneal inoculation of metacestode material containing protoscoleces (PSCs) obtained from infected plateau pikas were conducted on BALB/c mice. Furthermore, metacestode material without PSCs deriving from infected BALB/c mice was intraperitoneally inoculated to Mongolian jirds. Experimental animals were dissected for macroscopic and histopathological examination. The growth of cysts in BALB/c mice was infiltrative, and they invaded the murine entire body. Most of the metacestode cysts were multicystic, but a few were unilocular. The cysts contained sterile vesicles, which had no PSCs. The metacestode materials were able to successfully infect new mice. In the jirds model, E. shiquicus cysts were typically formed freely in the peritoneal cavity; the majority of these cysts were free while a small portion adhered loosely to nearby organs. The proportion of fertile cysts was high, and contained many PSCs. The PSCs produced in Mongolian jirds also successfully infected new ones, which confirms that jirds can serve as an alternative experimental intermediate host. In conclusion, a laboratory animal infection was successfully established for E. shiquicus using BALB/c mice and Mongolian jirds. These results provide new models for the in-depth study of Echinococcus metacestode survival strategy, host interactions and immune escape mechanism.

Past and present of diagnosis of echinococcosis: A review (1999-2021).

The larval forms of taeniid cestodes belonging to the genus Echinococcus are the source of the zoonotic infection known as echinococcosis. Alveolar and cystic echinococcosis are caused by Echinococcus multilocularis and Echinococcus granulosus (s. s), respectively. It is endemic in several regions of the world. In this systematic review, we describe diagnosis, and the species (human, canids, livestock, and small rodents) affected by cystic (CE) and alveolar echinococcosis (AE). From 1999 to 2021, we searched the online directory through PubMed, SCOPUS, Web of Science, and google scholar. Among the 37,700 records found in the online databases, 187 publications met our eligibility requirements. The majority of investigations employed a range of diagnostic methods, such as ELISA, imaging, copro-PCR, necropsy or arecoline hydrobromide purgation, morphological cestode confirmation, and fecal sieving/flotation to detect and confirm Echinococcus infection. ELISA was the most commonly used method followed by PCR, and imaging. The research team retrieved data describing the incidence or assessment of the diagnostic test for E. multilocularis in humans (N = 99), canids (N = 63), small ruminants (N = 13), large ruminants (N = 3), camel (N = 2), pigs (N = 2) and small mammals (N = 5). This study was conducted to explore the diagnostic tools applied to detect echinococcosis in humans as well as animals in prevalent countries, and to report the characteristic of new diagnostic tests for disease surveillance. This systematic review revealed that ELISA (alone or in combination) was the most common method used for disease diagnosis and diagnostic efficacy and prevalence rate increased when recombinant antigens were used. It is highly recommended to use combination protcols such as serological with molecular and imaging technique to diagnose disease. Our study identified scarcity of data of reporting echinococcosis in humans/ animals in low-income or developing countries particularly central Asian countries. Study reports in small rodents indicate their role in disease dissemination but real situation in these host is not reflected due to limited number of studies. Even though echinococcosis affects both public health and the domestic animal sector, therefore, it is important to devise new and strengthen implementation of the existing monitoring, judging, and control measures in this estimate.

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.

Phylogeny and population structure of Echinococcus granulosus (sensu stricto) based on full-length cytb-nad2-atp6 mitochondrial genes - First report from Sialkot District of Pakistan.

Cystic echinococcosis is a zoonotic disease of livestock having serious economic setbacks. The etiological agents of the disease belong to Echinococcus granulosus sensu lato. Despite of worldwide distribution of the disease, the molecular studies mainly employ amplification of cox1, nad1 and nad5 genes. To further strengthen the knowledge about significance of other molecular markers and to investigate the genetic diversity and population structure of Echinococcus species in Pakistan, the current study was designed in which full length mitochondrial cytb, atp6 and nad2 genes were amplified. Based on BLAST searches of the generated cytb, atp6 and nad2 gene sequences from a total of 18 hydatid cysts collected from cattle, 12 isolates were identified as E. granulousus G3 and 6 as E. granulosus (G1). The phylogeny inferred by the Bayesian method using nucleotide sequences of cytb-atp6-nad2 further confirmed their identity. The diversity indices indicated a high haplotype and a low nucleotide diversity. The negative values of Tajima's D and Fu's Fs test demonstrated deviation from neutrality suggesting a recent population expansion. To the best of our knowledge, the present study described the genetic variation of E. granulosus population for the first time in Pakistan using full-length cytb, atp6 and nad2 mitochondrial genes. The findings on the genetic variation of E. granulosus in Pakistan will constitute useful baseline information for future studies on the prevalence and population structure of E. granulosus based on full-length cytb, atp6 and nad2.

Prevalence, risk factors and first record of mitochondrial cox1 gene-based molecular characterization of Paramphistomum epiclitum from Pakistan.

Parasitic infestations are one of the major threats to the livestock industry in Pakistan. These have a negative impact on the production of domesticated livestock species. Paramphistomes belong to the superfamily Paramphistomoidea and are involved in infecting ruminants all over the world. To date, there was no information on mitochondrial DNA-based molecular characterization of Paramphistomum epiclitum from Pakistan. To close this research gap, this study was designed to provide insights into the epidemiology of Paramphistomum species. Paramphistomum epiclitum isolates were recovered from the rumen of small ruminants slaughtered at an abattoir located in Faisalabad city and animal demographics were recorded. DNA was extracted and mitochondrial cox1 was amplified and sequenced. Prevalence was calculated along with a 95% confidence interval in various groups. The chi-square test was applied to determine the association between different variables under investigation. A phylogenetic tree was constructed based on the Bayesian method. Population diversity indices were calculated using DnaSP 4.5 software. A total of 43 mutations were observed among 7 haplotypes. Negative values of Fu's Fs values, and Tajima's D indicated population expansion. Deworming, season, and grazing were the variables that significantly correlate (p < 0.05) with the prevalence of P. epiclitum. The high prevalence of P. epiclitum demonstrates that more studies are indeed needed to further understand the prevalence and distribution of P. epiclitum in definitive and all potential intermediate hosts in addition to intraspecies variation and relationship with populations from other locations.

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.

Comparison of mitochondrial genetic variation of Taenia hydatigena cysticerci from China and Mongolia.

Parasitic infection is one of the many challenges facing livestock production globally. Cysticercosis tenuicollis is a common parasitic disease in domestic and wild ruminants (intermediate host) caused by the larval stage of Taenia hydatigena that primarily infects dogs (definitive host). Although genetic studies on this parasite exist, only a few describe the genetic variation of this parasite in Mongolia. Our aim was thus, to identify the mitochondrial differences in ovine isolates of Cysticercus tenuicollis entering China from Mongolia and comparison with existing Chinese isolates from sheep and goats based on the recently described PCR-RFLP method and mitochondrial genes of NADH dehydrogenase subunit 4 (nad4) and the NADH dehydrogenase subunit 5 (nad5). Sixty-nine isolates were collected during routine veterinary meat inspections from sheep that originated from Mongolia, at the modern slaughterhouses in Erenhot City, Inner Mongolia. Additional 114 cysticerci were also retrieved from sheep and goats from northern (Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region, and Gansu Province), western (Tibet Autonomous Region), and southern (Jiangxi Province and Guangxi Province) China. The PCR-RFLP approach of the nad5 showed nine mitochondrial subclusters A1, A2, A3, A5, A8, A9, A10, A11, and B of T. hydatigena isolates from sheep and goats from Mongolia and China. Meanwhile, haplogroup A1 RFLP profile was more widespread than other variants. These data supplements existing information on the molecular epidemiology of T. hydatigena in China and Mongolia and demonstrate the occurrence of similar genetic population structures in both countries.

Herbal Medicines against Hydatid Disease: A Systematic Review (2000-2021).

Echinococcosis is a serious public health issue that affects people and livestock all over the world. Many synthetic and natural products have been examined in vitro and in vivo on Echinococcus species but only a few are used clinically, however, they may cause some complications and side effects. To overcome these limitations, new horizons of herbal drugs to cure echinococcosis are opening with every passing day. To summarize the developments during the last 21 years, we conducted this review of the literature to identify medicinal herbs utilized throughout the world that have anti-Echinococcus activity. From 2000 to 2021, data were carefully obtained from four English databases: Science Direct, PubMed, Scopus, and OpenGrey. Botanical name, extraction technique, extract quantities, efficacy, duration of treatment, year of publication, and half-maximal inhibitory concentration (IC50) values were all well noted. Ninety-one published papers, with 78 in vitro and 15 in vivo, fulfilled our selection criteria. Fifty-eight different plant species were thoroughly tested against Echinococcus granulosus. Zataria multiflora, Nigella sativa, Berberis vulgaris, Zingiber officinale (ginger), and Allium sativum were the most often utilized anti-Echinococcus herbs and the leaves of the herbs were extensively used. The pooled value of IC50 was 61 (95% CI 60−61.9) according to the random effect model and a large degree of diversity among studies was observed. The current systematic study described the medicinal plants with anti-Echinococcus activity, which could be investigated in future experimental and clinical studies to identify their in vivo efficacy, lethal effects, and mechanisms of action.

Understanding pathogen-host interplay by expression profiles of lncRNA and mRNA in the liver of Echinococcus multilocularis-infected mice.

Almost all Echinococcus multilocularis (Em) infections occur in the liver of the intermediate host, causing a lethal zoonotic helminthic disease, alveolar echinococcosis (AE). However, the long non-coding RNAs (lncRNAs) expression profiles of the host and the potential regulatory function of lncRNA during Em infection are poorly understood. In this study, the profiles of lncRNAs and mRNAs in the liver of mice at different time points after Em infection were explored by microarray. Thirty-one differentially expressed mRNAs (DEMs) and 68 differentially expressed lncRNAs (DELs) were found continuously dysregulated. These DEMs were notably enriched in "antigen processing and presentation", "Th1 and Th2 cell differentiation" and "Th17 cell differentiation" pathways. The potential predicted function of DELs revealed that most DELs might influence Th17 cell differentiation and TGF-ß/Smad pathway of host by trans-regulating SMAD3, STAT1, and early growth response (EGR) genes. At 30 days post-infection (dpi), up-regulated DEMs were enriched in Toll-like and RIG-I-like receptor signaling pathways, which were validated by qRT-PCR, Western blotting and downstream cytokines detection. Furthermore, flow cytometric analysis and serum levels of the corresponding cytokines confirmed the changes in cell-mediated immunity in host during Em infection that showed Th1 and Th17-type CD4+ T-cells were predominant at the early infection stage whereas Th2-type CD4+ T-cells were significantly higher at the middle/late stage. Collectively, our study revealed the potential regulatory functions of lncRNAs in modulating host Th cell subsets and provide novel clues in understanding the influence of Em infection on host innate and adaptive immune response.

The Multiplicity of Infection of Recombinant Vaccinia Virus Expressing the T7 RNA Polymerase Determines the Rescue Efficiency of Vesicular Stomatitis Virus.

Vesicular stomatitis virus (VSV) has a wide range of cell tropism, making it a prototype of studying the negative-strand RNA virus (NSRV), including virus-host interactions and vaccine development. Although VSV rescue systems have been progressively optimized throughout time, the T7-based expression system is the most commonly utilized to rescue VSV. However, it remains a significant barrier for many labs. In our study, we found that rescue VSV's efficiency is associated with the various multiplicities of infection (MOIs) of recombinant vaccinia virus expressing the T7 RNA polymerase (vTF-7.3). It works at maximum efficiency while the MOI of vTF-7.3 is 5, which is analyzed by quantitative PCR, Western blot, and flow cytometry, compared to the lowest rescue level with MOI of 1. Meanwhile, our data also suggest that purification of vTF-7.3 prior to transfection is a prerequisite for VSV rescue. Overall, our study reveals for the first time a precise correlation between vTF-7.3 and rescue efficiency, which may aid in resolving the uncertainties in the quest to build the VSV reverse genetic system.