Progression of asexual to sexual stages of Cystoisospora suis in a host cell-free environment as a model for Coccidia.
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ABSTRACT:
SUBMITTER: Feix AS
PROVIDER: S-EPMC8426156 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:The porcine coccidium Cystoisospora suis is characterized by a complex life-cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Dynein light chain type 1 and male gamete fusion factor HAP2 are restricted to microgametes. Tyrosine-rich proteins and oocyst wall proteins are a part of the oocyst wall. The Rad51/Dmc1-like protein and Nima-related protein kinases are associated with the cell cycle and fertilization process. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development. METHODS:Sexual stages of C. suis developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Transcription levels were compared for parasites in culture supernatants at 6-9 days of cultivation (doc) and intracellular parasites at 6-15 doc. RESULTS:Sexual stage of C. suis was detected during 8-11 doc in vitro. Microgamonts (16.8?±?0.9 µm) and macrogamonts (16.6?±?1.1 µm) are very similar in shape and size. Microgametes had a round body (3.5?±?0.5 µm) and two flagella (11.2?±?0.5 µm). Macrogametes were spherical with a diameter of 12.1?±?0.5 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. CONCLUSIONS:The present study linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in this unique model for coccidian parasites. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of C. suis development (applicable for coccidian parasites in general) which could be exploited as a novel target for control.
| S-EPMC7079422 | biostudies-literature
Project description:The apicomplexan parasite Cystoisospora suis has global significance as an enteropathogen of suckling piglets. Its intricate life cycle entails a transition from an asexual phase to sexual development, ultimately leading to the formation of transmissible oocysts. To advance our understanding of the parasite’s cellular development, we complemented previous transcriptome studies by delving into the proteome profiles at five distinct time points of in vitro cultivation through LC/MS-MS analysis. A total of 1324 proteins were identified in the in vitro developmental stages of C. suis, and 1082 proteins were identified as significantly differentially expressed. We performed BLAST, GO enrichment, and KEGG pathway analyses on the up- and downregulated proteins to elucidate correlated events in the C. suis life cycle. Our analyses revealed intriguing metabolic patterns in macromolecule metabolism, DNA and RNA-related processes, proteins associated with sexual stages, and those involved in cell invasion, reflecting the adaptation of sexual stages to a nutrient-poor and potentially stressful extracellular environment, with a focus on enzymes involved in metabolism and energy production. These findings have important implications for understanding the developmental biology of C. suis as well as other, related coccidian parasites, such as Eimeria spp. and Toxoplasma gondii. They also support the role of C. suis as a new model for the comparative biology of coccidian tissue cyst stages.
2023-10-13 | PXD045050 | Pride
Project description:Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (?1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.
| S-EPMC3702598 | biostudies-literature
Project description:After introduction of the anticoccidial toltrazuril for the metaphylactic treatment of suckling piglet coccidiosis, only few field evaluations on the effect of treatment against the causative agent, Cystoisospora suis, were performed. In 2018, a field study was conducted to detect the presence of the parasite on pig farms in four different European countries, and to evaluate management parameters possibly associated with infection and disease. A total of 49 farms from Austria, the Czech Republic, Germany and Spain were included. Repeated pooled fecal samples from 603 litters were taken in the 2nd and 3rd week of life. Samples were examined by autofluorescence for the presence of C. suis, and fecal consistency was scored. For each farm a questionnaire was provided to document management and treatment history. Feces scored as diarrhoeic were not significantly more often positive for C. suis than non-diarrhoeic feces but samples from litters with previously reported occurrence of diarrhea were significantly more often positive (p = 0.000). Pasty feces were significantly more often positive than those of other consistency (p = 0.005). Overall, 71.4% of the farms and 50.1% of the litters were positive for C. suis at least once. The prevalence on the farms reached up to 100%. Diarrhea was seen in samples from 53.1% of the farms (9.6% of the litters). Cystoisospora suis was diagnosed on 80.8% of the farms with vs. 60.8% of those without diarrhea. Toltrazuril was applied on 30 farms, and of these 53.3% had diarrhoeic samples and 66.7% were positive for C. suis vs. 19 farms that did not use toltrazuril with 52.6% diarrhoeic and 79.0% C. suis positive samples (p > 0.05). Only on two farms a disinfectant with activity against coccidia was used, and C. suis was not detected there. Current control of C. suis appears to be insufficient on the majority of the examined farms. These findings highlight the importance of correct application of medication, and an effective hygiene management. To maintain effective parasite control, efficacy monitoring of the control measures should be implemented.
| S-EPMC7064465 | biostudies-literature
Project description:Earlier genetic and inhibitor studies showed that epigenetic regulation of gene expression is critical for malaria parasite survival in multiple life stages and a promising target for new antimalarials. We therefore evaluated the activity of 350 diverse epigenetic inhibitors against multiple stages of Plasmodium falciparum We observed ≥90% inhibition at 10 μM for 28% of compounds against asexual blood stages and early gametocytes, of which a third retained ≥90% inhibition at 1 μM.
| S-EPMC7318029 | biostudies-literature
Project description:Here we report one of the first investigations of evolvability of lifespan and reproduction in metazoans, examining both extrinsic and intrinsic factors. We tested effects on senescence of an environmental variable (simulated lake hydroperiod, the length of time an aquatic habitat is inundated), female reproductive physiology (asexual females that reproduce by ameiosis, versus sexual females reproducing by meiosis), and time in a benign culture environment (minimal, if any, external mortality factors). To do this we established chemostat cultures of the rotifer Brachionus plicatilis s.s., and maintained the cultures for 385 d. Hydroperiod alone or in interaction with the effects of time in the benign environment (season) or reproductive physiology had no significant effect on the net reproductive rate, generation time, or rate of aging. Yet combining animals from both ephemeral and permanent hydroperiods revealed a 26% increase in asexual female lifespan across seasons (23% decrease in the rate of aging) and a 56% increase in asexual fecundity, suggesting that maintenance in benign laboratory conditions leads to slower aging. The relative stasis of traits for sexual females implies an impact of reproductive physiology on evolvability. In addition we found a positive correlation between fecundity and lifespan, suggesting an absence of trade-offs in life history traits in the benign laboratory environment.
| S-EPMC4004349 | biostudies-literature
Project description:BackgroundConstant treatment regimens with toltrazuril against Cystoisospora suis infection in piglets are being applied in the intensive production systems for the last two decades, but the possibility of resistance development has not been addressed so far despite limited availability of treatment alternatives. Recently, a pig producer in The Netherlands who routinely used toltrazuril complained about diarrhea in suckling piglets in the absence of bacterial and viral pathogens, and oocysts of C. suis could be isolated from feces of affected litters.MethodsPiglets from two litters were infected with a field isolate of C. suis, Holland-I, and treated with 0 (Holl-Ctrl), 20 (Holl-20) or 30 (Holl-30) mg/kg of body weight (BW) of toltrazuril (Baycox®). The efficacy of toltrazuril was measured by assessment of oocyst excretion, fecal consistency and BW gain. A separate litter was infected with a toltrazuril-susceptible strain of C. suis, Wien-I, and treated with 0 (Wien-Ctrl) or 20 (Wien-20) mg/kg BW of toltrazuril for comparison.ResultsTreatment with the recommended (20 mg/kg) dose of toltrazuril completely suppressed oocyst shedding and diarrhea in group Wien-20. The prevalence of oocyst excretion was 100% in the groups infected with Holland-I and 80% in the group Wien-Ctrl. Most days with diarrhea were observed in group Holl-20 with an average of 6.40%, followed by 5.71% in Wien-Ctrl, while in Holl-Ctrl and Holl-30 diarrhea was only seen in 1.79% of the samples (n = 14/piglet). Oocyst excretion, fecal consistency and BW gain did not differ significantly among groups infected with Holland-I, indicating loss of efficacy to toltrazuril.ConclusionExperimental infections and treatment confirmed toltrazuril resistance against the field isolate even at increased dosage. Such isolates are a potential threat to pig production as no other effective and economically sustainable alternative treatment is currently available. In the absence of a standardized protocol for resistance testing in C. suis, regular parasitological examination and, if possible, experimental confirmation should be considered to evaluate the extent and consequences of toltrazuril resistance.
| S-EPMC5492287 | biostudies-literature
Project description:BACKGROUND: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved. RESULTS: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13C-glucose and 13C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death. CONCLUSIONS: Our metabolomics approach has allowed us to revise current models of P. falciparum carbon metabolism. In particular, we found that both asexual and sexual blood stages utilize a conventional TCA cycle to catabolize glucose and glutamine. Gametocyte differentiation is associated with a programmed remodeling of central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector. The increased sensitivity of gametocyte stages to TCA-cycle inhibitors provides a potential target for transmission-blocking drugs.
| S-EPMC3704724 | biostudies-literature
Project description:It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown.We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized.It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.
| S-EPMC3266614 | biostudies-literature
Project description:BackgroundTo date, investigations on the immune response to Cystoisospora suis infections focused on suckling piglets, the age group clinically most affected. Actively immunizing piglets is unfeasible due to their immature immune system and the typically early infection in the first days after birth. Therefore, understanding and possibly enhancing the immune response of immune-competent animals is the prerequisite to develop a passive immunization strategy for piglets which currently rely on very limited treatment options.MethodsTo investigate antibody and cytokine responses of immune-competent animals and the impact of the oral immunization protocol on their immune response, growers with unknown previous exposure to C. suis (10-11 weeks-old) were infected one or three times with different doses (600 and 6000 or 200 and 2000, respectively) of C. suis oocysts, and compared to uninfected controls. Oocyst excretion was evaluated, and blood and intestinal mucus antibody titers were determined by IFAT. Systemic production of Th1, Th2, inflammatory and regulatory cytokines was determined in different immune compartments at mRNA and (after stimulation with a recombinant merozoite-protein) at protein level by PCR and multiplex fluorescent immunoassay, respectively.ResultsInfection generated significantly increased serum IgA and IgG levels against C. suis sporozoites and merozoites, irrespective of infection mode, with IgG against merozoites showing the strongest increase. No clinical signs and only occasional excretion were observed. The systemic cytokine response to C. suis was only weak. Nonetheless, in white blood cells, IL-4, IL-6 and IL-10 mRNA-levels significantly increased after infection, whereas IFN-?, IL-2 and TGF-? expression tended to decrease. In mesenteric lymph nodes (MLN), IL-10 and TNF-? levels were elevated while splenic cytokine expression was unaltered upon infection. Stimulated MLN-derived lymphocytes from infected pigs produced slightly more IL-12 and less IFN-? than controls.ConclusionsAn infection and a subsequent systemic immune response can be induced in immune-competent animals by all evaluated infection models and growers can be used as models to mimic sow immunizations. The immune response to C. suis, although mild and with considerable variation in cytokine expression, was characterized by a Th2-associated and regulatory cytokine profile and antibody production. However, none of the parameters clearly stood out as a potential marker associated with protection. Antibody titers were significantly positively related with oocyst excretion and might thus serve as correlates for parasite replication or severity of infection.
| S-EPMC6031197 | biostudies-literature