Protein disulfide isomerase PDI8 is indispensable for parasite growth and associated with secretory protein processing in Toxoplasma gondii.

Protein disulfide isomerase, containing thioredoxin (Trx) domains, serves as a vital enzyme responsible for oxidative protein folding (the formation, reduction, and isomerization of disulfide bonds in newly synthesized proteins) in the endoplasmic reticulum (ER). However, the role of ER-localized PDI proteins in parasite growth and their interaction with secretory proteins remain poorly understood. In this study, we identified two ER-localized PDI proteins, TgPDI8 and TgPDI6, in Toxoplasma gondii. Conditional knockdown of TgPDI8 resulted in a significant reduction in intracellular proliferation and invasion abilities, leading to a complete block in plaque formation on human foreskin fibroblast monolayers, whereas parasites lacking TgPDI6 did not exhibit any apparent fitness defects. The complementation of TgPDI8 with mutant variants highlighted the critical role of the CXXC active site cysteines within its Trx domains for its enzymatic activity. By utilizing TurboID-based proximity labeling, we uncovered a close association between PDI proteins and canonical secretory proteins. Furthermore, parasites lacking TgPDI8 showed a significant reduction in the expression of secretory proteins, especially those from micronemes and dense granules. In summary, our study elucidates the roles of TgPDI8 and sets the stage for future drug discovery studies. IMPORTANCE: Apicomplexans, a phylum of intracellular parasites, encompass various zoonotic pathogens, including Plasmodium, Cryptosporidium, Toxoplasma, and Babesia, causing a significant economic burden on human populations. These parasites exhibit hypersensitivity to disruptions in endoplasmic reticulum (ER) redox homeostasis, necessitating the presence of ER-localized thioredoxin (Trx) superfamily proteins, particularly protein disulfide isomerase (PDI), for proper oxidative folding. However, the functional characteristics of ER-localized PDI proteins in Toxoplasma gondii remain largely unexplored. In this study, we identified two ER-localized proteins, namely, TgPDI8 and TgPDI6, and demonstrated the indispensable role of TgPDI8 in parasite survival. Through a comprehensive multi-omics analysis, we elucidated the crucial role of TgPDI8 in the processing of secretory proteins in T. gondii. Additionally, we introduced a novel ER-anchored TurboID method to label and identify canonical secretory proteins in T. gondii. This research opens up new avenues for understanding oxidative folding and the secretory pathway in apicomplexan parasites, laying the groundwork for future advancements in antiparasitic drug development.

Transcriptomic analysis uncovers a biphasic response to precocious Eimeria acervulina infection in chicken duodenal tissue.

Live anticoccidial vaccines, either formulated with unattenuated or attenuated Eimeria parasites, are powerful stimulators of chicken intestinal immunity. Little is known about the dynamics of gene expression and the corresponding biological processes of chicken responses against infection with precocious line (PL) of Eimeria parasites. In the present study, we performed a time-series transcriptomic analysis of chicken duodenum across 15 time points from 6 to 156 hours post-infection (p.i.) with PL of E. acervulina. A high-quality profile showing two distinct changes in chicken duodenum mRNA expression was generated during the infection of Eimeria. Early response revealed that activation of the chicken immune response was detectable from 6 h.p.i., prominent genes triggered during the initiation of asexual and sexual parasite growth encompass immune regulatory effects, such as interferon gamma (IFN-γ), interferon regulatory factor 1 (IRF1), and interleukin-10 (IL10). The late response was identified significantly associating with maintaining cellular structure and activating lipid metabolic pathways. These analyses provide a detailed depiction of the biological response landscape in chickens infected by the PL of E. acervulina, contributing significant insights for the investigation of the host-parasite interactions and the management of parasitic diseases.

Transcriptomic Insights into the Developmental Dynamics of Eimeria acervulina: A Comparative Study of a Precocious Line and the Wild Type.

Coccidiosis, a parasitic disease caused by single or multiple Eimeria species, leads to significant economic losses in the poultry industry. The Eimeria life cycle includes schizogony, gametogony, and sporogony. To investigate the dynamics of gene expression and regulatory networks during the development of Eimeria acervulina, we employed time-course transcriptomics to rigorously compare the gene expression patterns between a precocious line (PL) and the wild type (WT) of E. acervulina. The results revealed that the PL enters into gametogony 12 h earlier than the WT, and both the PL and WT exhibited distinct clustering patterns during the development phase. A weighted gene co-expression network analysis (WGCNA) identified genes specifically expressed at four distinct developmental stages, schizogony, gametogony, sporulated oocysts, and unsporulated oocysts, clarifying the key biological processes at each stage. This study used global transcriptome profiling to elucidate molecular variations throughout the E. acervulina life cycle, providing critical insights into molecular characterization and valuable resources for investigating other apicomplexan parasites of public health importance.

Study on the effects of toosendanin against Eimeria tenella and its impact on the apoptosis of cecal cells in chicks.

To investigate the therapeutic effect of toosendanin (TSN) against Eimeria tenella (E. tenella) in chicks. In this experiment, a chick model of artificially induced E. tenella infection was established. The anti-coccidial effect was investigated by treating different doses of TSN. A preliminary mechanism of action was conducted, using cecal cell apoptosis as a starting point. TSN at the concentration of 5 mg/kg BW showed the best effect against E. tenella with the ACI value of 164.35. In addition, TSN reduced pathological damage to cecal tissue, increased the secretion of glycogen and mucus in cecal mucosa, and enhanced the mucosal protective effect. It also elevated the levels of IFN-γ, IL-2, and IgG in serum, and raised the sIgA content in cecal tissue of infected chicks, thereby improving overall immune function. TSN was observed to promote the apoptosis of cecum tissue cells by TUNEL staining analysis. Immunohistochemistry analysis revealed that in TSN-treated groups, the expression of Caspase-3 and Bax was elevated, while the expression of Bcl-2 was reduced. TSN induced apoptosis in host cells by dose-dependently decreasing the Bcl-2/Bax ratio and upregulating Caspase-3 expression. In summary, TSN exhibited significant anticoccidial efficacy by facilitating apoptosis in host cecal cells, with the most pronounced effect observed at a dosage of 5 mg/kg body weight.

Oral delivery of Eimeria acervulina transfected sequentially with two copies of the VP2 gene induces immunity against infectious bursal disease virus in chickens.

Chicken coccidiosis caused by Eimeria spp. can occur on almost all poultry farms, causing huge economic losses to the industry. Genetically manipulated Eimeria parasites as a vaccine vector to deliver viral antigens have been reported. In our preliminary study, transgenic E. acervulina expressing a VP2 gene (Ea-VP2) of the infectious bursal disease virus (IBDV) demonstrated partial protection against IBDV infection. To enhance immune responses, we aimed to increase the VP2 gene copy number in transgenic E. acervulina. In this study, we used a novel plasmid vector carrying a VP2 gene fused with three flag tags and a red fluorescent reporter gene (mCherry). The vector was introduced into Ea-VP2 sporozoites through nucleofection, leading to the generation of Ea-2VP2. Subsequent analysis revealed a notable escalation in the fluorescent rate, increasing from 0.11 to 95.1% following four consecutive passages facilitated by fluorescent-activated cell sorting. Verification via PCR, Western blot, and immunofluorescence confirmed the successful construction of the Ea-2VP2 population. Despite lower fecundity compared to wild-type E. acervulina, Ea-2VP2 maintained immunogenicity. Our research effectively created a transgenic E. acervulina strain transfected sequentially with two copies of the VP2 gene from IBDV. This modification resulted in an increased humoral immune response after primary immunization in chickens. Additionally, it demonstrated a degree of protection within the bursa against IBDV infection. Future studies will focus on further enhancing immune response levels.

Transcriptomic responses of rabbits to infections by precocious line and wild-type Eimeria media: revealing molecular signatures and pathway differences in liver and duodenum during the peak and terminal phases of oocyst production.

Eimeria media is a principal pathogen responsible for rabbit coccidiosis, targeting the rabbit's intestinal epithelial cells. This parasitism damages the intestinal mucosal barrier, initiating a systemic immune and inflammatory response that jeopardizes the sustainable growth of rabbit farming. To understand the implications of infection on the host's immune and metabolic responses, we employed RNA-Seq to analyze RNA from the liver and duodenum tissues of post-infected rabbits infected with both the precocious line and wild-type strain of E.media. Comprehensive transcriptomic analysis revealed that the two parasites exhibit divergent transcriptomic imprints on host tissues. While the precocious line predominantly modulates immune-centric pathways with significant differential gene enrichment, wild-type strain favors pathways that affect metabolism. In addition, our study pinpointed a set of genes that undergo significant modifications in response to these effects. These revelations grant a fresh avenue to probe deeper into the symbiotic intricacies of the E.media and its rabbit host.

Anthelmintic efficacy of nitazoxanide in dogs naturally infected with Toxocara canis.

Toxocara canis (T. canis) is a gastrointestinal nematode in dogs, and its larvae also infect humans, causing severe larval migratory disease. Anthelmintic drugs have become the primary means to combat T. canis. In this study, the efficacy of nitazoxanide (NTZ) was tested against all the internal stages of T. canis, including L3 larval stage in vitro experiments and gastrointestinal worm in vivo experiments. In the in vitro experiment, after treatment with NTZ at 7.81 and 62.5 µg/mL for 12 h, the larval mortality efficacy reached 90.0 and 100.0%, respectively. In the in vivo experiments, 100 mg/kg NTZ possessed good anthelmintic efficacy against T. canis, with an egg per gram (EPG) reduction of 99.19%, and 90.00% of dogs cleared with residual worms. These results were comparable to those of the positive control drug. The highest anthelmintic efficacy was observed in the group treated with 150 mg/kg NTZ. Based on faecal egg counts, the number of T. canis eggs decreased by 100.00%, and the percentage of dogs cleared with residual worms achieved 90.00% after 7 days of treatment in the 150-mg/kg NTZ treatment group. In general, NTZ showed great potential to be applied as an anthelmintic against T. canis.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites.

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Ponazuril: Clinical efficacy, ultrastructure, and histopathology studies of in vivo anticoccidial action against Eimeria tenella.

Ponazuril, a novel antiprotozoal drug in the class of triazine, has shown a promising application on apicomplexan infections in poultry and livestock. However, the effect and mechanism of action of ponazuril against Eimeria tenella (E. tenella) are unclear. The efficacy against E. tenella was initially studied by administering different doses of ponazuril in drinking water. The treated stage and site of ponazuril on E. tenella were observed through ultrastructural and histopathological analyses. Chicks were orally treated with a dose of 15 mg/kg body weight of ponazuril at different endogenous stages of E. tenella post-infection. According to the clinical study, the values of anticoccidial indices (ACI) were 157.0, 162.3, 196.9, 194.5, and 190.9, respectively, when the ponazuril was administered in drinking water at doses of 5, 10, 20, 40, and 50 mg/L for two consecutive days after infection. Among them, the 20 mg/L ponazuril group showed the best anticoccidial effect, which was superior to that of the toltrazuril treatment group, with an ACI value of 191.7. Histological analysis indicated that ponazuril effectively relieved cecal lesions, and decreased the number of merozoites. Transmission electron micrographs (TEM) observed that merozoites became irregular in shape, and some apparent protrusions of the outer membrane were presented especially the second-generation merozoites. Additionally, abnormalities in the development of WFBI and WFBII in the macrogametocyte were observed, which may affect the formation of the ovule wall. Moreover, merozoites in the treated group showed uneven and marginalized chromatin and mitochondrial swelling. These results suggested ponazuril is a potential anticoccidial drug, providing information on the mechanism of anticoccidial effects.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella.

BACKGROUND: Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. METHODS: We developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule. RESULTS: We obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate. CONCLUSIONS: Our findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.

Live attenuated anticoccidial vaccines for chickens.

Chicken coccidiosis, caused by infection with single or multiple Eimeria species, results in significant economic losses to the global poultry industry. Over the past decades, considerable efforts have been made to generate attenuated Eimeria strains, and the use of live attenuated anticoccidial vaccines for disease prevention has achieved tremendous success. In this review, we evaluate the advantages and limitations of the methods of attenuation as well as attenuated Eimeria strains in a historical perspective. Also, we summarize the recent exciting research advances in transient/stable transfection systems and clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing developed for Eimeria parasites, and discuss trends and challenges of developing live attenuated anticoccidial vaccines based on transgenesis and genome editing.

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony.

BACKGROUND: The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood. METHODS: In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development. RESULTS: In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output. CONCLUSIONS: The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites.

Matrine provides a protective effect against Eimeria tenella challenge by alleviating intestinal barrier damage.

As a virulent and harmful protozoan, Eimeria tenella (E.tenella) causes harmful chicken coccidiosis, inducing high economic losses in the chicken industry. The management of the coccidial disease has been greatly hampered by drug resistance. Matrine is an active ingredient from Ku Shen (Radix Sophorae Flavescentis), a typical pesticide in chinese medicine. The aim of this study was to examine matrine's possible effectiveness in the treatment of coccidiosis and its protective function on the intestinal barrier. The anticoccidial index (ACI), the levels of anti-oxidant indexes, and secretory immunoglobulin A (sIgA) were detected. The levels of mRNA and protein expression of Occludin, ZO-1, and Claudin-1 were determined through quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) analysis. Matrine exhibited a moderate ACI value, and ACI values of 122.51 and 143.42 corresponded to 5 and 10 mg/kg of matrine, respectively. Compared to the infective control group, the expression of tight junction proteins significantly increased in the matrine-treatment group by RT-PCR and IHC analysis, which are essential for the mucosal immune system and the intestinal barrier. Besides, the matrine-treatment group showed a more complete intestinal structure, fewer bleeding spots, and coccidian by histopathology analysis. We also found that, matrine significantly enhanced the antioxidant ability and significantly increased the content of sIgA. Above all, matrine was considered an efficient drug against E.tenella by the anti-oxidant efficacy, and the ability to protect the composition and function of the intestinal barrier.

Forward genetic analysis of monensin and diclazuril resistance in Eimeria tenella.

Worldwide distributed coccidiosis is caused by infection of both Eimeria species and Cystoisospora in the host intestine and causes huge economic losses to the livestock industry, especially the poultry industry. The control of such diseases relies mainly on chemoprophylaxis with anticoccidials, which has led to a very common drug resistance in this field. However, the genetic mechanisms underlying resistance to many anticoccidial drugs remain unknown. In this study, strains of E. tenella resistant to 250 mg/kg monensin were generated and characterized. Forward genetic approaches based on pooled genome sequencing, including experimental evolution and linkage group selection, were used to locate candidate targets responsible for resistance to monensin and diclazuril in E. tenella. A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains, and two genomic regions with strong selection signals were also detected in diclazuril-resistant strains. Our study reveals the genetic characterization of the experimental evolution and linkage group selection in Eimeria species, and also provides important information that contributes to the understanding of the molecular mechanism of drug resistance in coccidia.

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella.

The antiparasitic drug halofuginone is important for controlling apicomplexan parasites. However, the occurrence of halofuginone resistance is a major obstacle for it to the treatment of apicomplexan parasites. Current studies have identified the molecular marker and drug resistance mechanisms of halofuginone in Plasmodium falciparum. In this study, we tried to use transcriptomic data to explore resistance mechanisms of halofuginone in apicomplexan parasites of the genus Eimeria (Apicomplexa: Eimeriidae). After halofuginone treatment of E. tenella parasites, transcriptome analysis was performed using samples derived from both resistant and sensitive strains. In the sensitive group, DEGs associated with enzymes were significantly downregulated, whereas the DNA damaging process was upregulated after halofuginone treatment, revealing the mechanism of halofuginone-induced parasite death. In addition, 1,325 differentially expressed genes (DEGs) were detected between halofuginone resistant and sensitive strains, and the DEGs related to translation were significantly downregulated after halofuginone induction. Overall, our results provide a gene expression profile for further studies on the mechanism of halofuginone resistance in E. tenella.

Tissue-resident, memory CD8+ T cells are effective in clearing intestinal Eimeria falciformis reinfection in mice.

Eimeria, a cousin of malarial parasites, causes coccidiosis that results in huge losses in the poultry industry. Although live coccidiosis vaccines have been developed and used widely for the successful control of the disease, the mechanism underlying protective immunity remains largely unknown. Using Eimeria falciformis as a model parasite, we observed that tissue-resident memory CD8+ T (Trm) cells accumulated in cecal lamina propria following E. falciformis infection in mice, especially after reinfection. In convalescent mice challenged with a second infection, E. falciformis burden diminished within 48-72 h. Deep-sequencing revealed that CD8+ Trm cells were characterized by rapid up-regulation of effector genes encoding pro-inflammatory cytokines and cytotoxic effector molecules. While FTY720 (Fingolimod) treatment prevented the trafficking of CD8+ T cells in peripheral circulation and exacerbated primary E. falciformis infection, such treatment had no impact on the expansion of CD8+ Trm cells in convalescent mice receiving secondary infection. Adoptive transfer of cecal CD8+ Trm cells conferred immune protection in naïve mice, indicating that these cells provide direct and effective protection against infection. Overall, our findings not only explain a protective mechanism of live oocyst-based anti-Eimeria vaccines but also provide a valuable correlate for assessing vaccines against other protozoan diseases.

Study on the pathogenicity, immunogenicity, endogenous development and drug sensitivity of Eimeria kongi.

Following the discovery of Eimeria kongi, we investigated the pathogenicity, immunogenicity, endogenous development and drug sensitivity of this coccidian. Coccidia-free rabbits were inoculated with 1 × 102 to 5 × 104 sporulated oocysts of E. kongi before challenge 14 days post inoculation. E. kongi was moderately pathogenic and induced good immunity against re-infection. All inoculated doses results in reduced food intake and body weight gain, and an inoculation oocyst dose of 1 × 103 or higher caused various degrees of diarrhea. Except for one death of the highest dose group, all rabbits recovered 12 days post inoculation. An inoculation dose of 1 × 103 or 1 × 104 oocysts conferred the most effective protection from re-infection, which reduced oocyst output by approximately 99% and maintained body weight gain. Four generations of schizogony were observed, and the endogenous development mainly occurred in the jejunum and ileum of rabbits. E. kongi was most sensitive to sulfachloropyrazine sodium, followed by decoquinate; it is resistant to diclazuril. Both decoquinate and sulfachloropyrazine sodium may be effective in the control of E. kongi infection.

EtcPRS Mut as a molecular marker of halofuginone resistance in Eimeria tenella and Toxoplasma gondii.

The control of coccidiosis, causing huge economic losses in the poultry industry, is facing the stagnation of the development of new drugs and the emergence of drug resistance. Thus, the priority for coccidiosis control is to decipher the effect mechanisms and resistance mechanisms of anticoccidial drugs. In this study, we mined and validated a molecular marker for halofuginone resistance in Eimeria tenella through forward and reverse genetic approaches. We screened whole-genome sequencing data and detected point mutations in the ETH2_1020900 gene (encoding prolyl-tRNA synthetase, PRS). Then, we introduced this mutated gene into E. tenella and Toxoplasma gondii and validated that overexpression of this mutated gene confers resistance to halofuginone in vivo and in vitro. These results together show that mutations A1852G and A1854G on the ETH2_1020900 gene are pivotal to halofuginone resistance in E. tenella, encouraging the exploration of mechanisms of drug resistance against other anticoccidial drugs in eimerian parasites.

Evidence of high-efficiency cross fertilization in Eimeria acervulina revealed using two lines of transgenic parasites.

Eimeria species are apicomplexan parasites with a direct life cycle consisting of a replicative phase involving multiple rounds of asexual replication in the intestine or other organs including kidneys, liver, and gallbladder, depending on the species, followed by a sexual phase or gamogony involving the development and fertilization of gametes, an essential process for Eimeria transmission. Recent advances in the genetic manipulation of these parasites made it possible to conduct genetic crosses combined with genomic approaches to elucidate the genetic determinants of Eimeria development, virulence, drug resistance, and immune evasion. Here, we employed genetic techniques to generate two transgenic Eimeria acervulina lines, EaGAM56 and EaHAP2, each expressing two unique fluorescent proteins, with one controlled by a constitutive promotor for cross-efficiency analysis and the other by a male or female gametocyte stage-specific promoter to observe sexual development. The expression of fluorescent proteins in the transgenic lines was analyzed in different developmental stages of the E. acervulina life cycle by immunoblotting and by examination of frozen sections using fluorescence microscopy. The effect of infective doses on cross-fertilization was further investigated by conducting several genetic crosses between the two transgenic lines at different doses and ratios. Two transgenic lines expressing constitutive and gametocyte-specific fluorescence proteins were generated and characterized. These transgenic parasites display synchronous development in chickens, comparable with that of the wild type. Genetic crosses between the two transgenic parasites showed that a high rate of oocysts co-expressing the two reporters could be achieved following inoculation with high doses of infective oocysts. We further showed that the proportion of co-transfected oocysts can be modulated by altering the ratio of the transgenic parental lines. Higher infective doses and similar numbers of functional gametocytes from the parents increase the rate of cross-fertilization. Our data highlight the usefulness of genetic manipulation and fluorescently-labeled transgenic gametocytes as tools to study Eimeria development and to elucidate the factors that modulate sexual development. This work sets the stage for the implementation of novel approaches to investigate other aspects of Eimeria pathogenesis, virulence, and drug susceptibility and resistance.