Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca2+) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

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A Comparative Genomic Study of Attenuated and Virulent Strains of Babesia bigemina

Pathogens ◽

10.3390/pathogens10030318 ◽

2021 ◽

Vol 10 (3) ◽

pp. 318

Author(s):

Bernardo Sachman-Ruiz ◽

Luis Lozano ◽

José J. Lira ◽

Grecia Martínez ◽

Carmen Rojas ◽

...

Keyword(s):

In Vitro Culture ◽

Virulence Genes ◽

Laboratory Strain ◽

Host Cells ◽

Comparative Genomic ◽

Local Alignment ◽

Host Immune System ◽

Babesia Bigemina ◽

Genomic Study

Cattle babesiosis is a socio-economically important tick-borne disease caused by Apicomplexa protozoa of the genus Babesia that are obligate intraerythrocytic parasites. The pathogenicity of Babesia parasites for cattle is determined by the interaction with the host immune system and the presence of the parasite’s virulence genes. A Babesia bigemina strain that has been maintained under a microaerophilic stationary phase in in vitro culture conditions for several years in the laboratory lost virulence for the bovine host and the capacity for being transmitted by the tick vector. In this study, we compared the virulome of the in vitro culture attenuated Babesia bigemina strain (S) and the virulent tick transmitted parental Mexican B. bigemina strain (M). Preliminary results obtained by using the Basic Local Alignment Search Tool (BLAST) showed that out of 27 virulence genes described and analyzed in the B. bigemina virulent tick transmitted strain, only five were fully identified in the attenuated laboratory strain. In all cases, the identity and coverture of the identified genes of the wildtype strain were higher than those of the laboratory strain. This finding is putatively associated with the continuous partial loss of virulence genes in the laboratory strain after several passages of the parasite population under optimal in vitro growth conditions. The loss of virulence factors might be reflected in the absence of symptoms of the disease in cattle inoculated with the attenuated strain despite the presence of infection in the bovine host cells.

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Eimeria tenella: Vitamin Requirements for Development in Primary Cultures of Chicken Kidney Cells

The Journal of Protozoology ◽

10.1111/j.1550-7408.1978.tb04183.x ◽

1978 ◽

Vol 25 (4) ◽

pp. 544-546 ◽

Cited By ~ 3

Author(s):

D. J. DORAN ◽

P. C. AUGUSTINE

Keyword(s):

Primary Cultures ◽

Eimeria Tenella ◽

Kidney Cells ◽

Vitamin Requirements ◽

Chicken Kidney

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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

10.21203/rs.3.rs-58018/v1 ◽

2020 ◽

Author(s):

Carlos Suarez ◽

Marta G. Silva ◽

Reginaldo G. Bastos ◽

J. Stone Doggett ◽

Michael K. Riscoe ◽

...

Keyword(s):

Mononuclear Cells ◽

Babesia Bovis ◽

Host Cells ◽

Bovine Babesiosis ◽

Theileria Equi ◽

Peripheral Blood Mononuclear ◽

Equine Piroplasmosis ◽

Apicomplexan Parasites ◽

And Control

Abstract Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. Endochin-like quinolones (ELQ)-300 and ELQ-316 proved safe and efficacious against related apicomplexans, such as Plasmodium spp., and ELQ-316 was also effective against B. microti, without showing toxicity in mammals.Methods: Inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. Percentage of parasitized erythrocytes was measured by flow cytometry. Effect of the ELQ drugs on the viability of actively replicating horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by ELISA. Results: We calculated IC50 ranging from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 at 72 hr among all cultured parasites tested. None of the parasites tested were able to replicate in cultures in the presence of the ELQs-300 and ELQ-316 at IC100, which range from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 hours. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions: ELQ-300 and ELQ-316 have a significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

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Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

International Journal of Molecular Sciences ◽

10.3390/ijms23010068 ◽

2021 ◽

Vol 23 (1) ◽

pp. 68

Author(s):

Izra Abbaali ◽

Danny A. Truong ◽

Shania D. Day ◽

Nancy Haro-Ramirez ◽

Naomi S. Morrissette

Keyword(s):

Toxoplasma Gondii ◽

Host Cells ◽

Proof Of Concept ◽

Systematic Analysis ◽

Apicomplexan Parasites ◽

Microtubule Disruption ◽

Antiparasitic Drugs ◽

General Response ◽

Babesia Spp

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

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Multiparameter Optimization of Trypanocidal Cruzain Inhibitors With In Vivo Activity and Favorable Pharmacokinetics

Frontiers in Pharmacology ◽

10.3389/fphar.2021.774069 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ivani Pauli ◽

Celso de O. Rezende Jr. ◽

Brian W. Slafer ◽

Marco A. Dessoy ◽

Mariana L. de Souza ◽

...

Keyword(s):

Parasite Burden ◽

Host Cells ◽

Benzimidazole Derivatives ◽

Pharmacokinetic Studies ◽

Host Immune System ◽

Novel Drugs ◽

Main Metabolic Pathway ◽

Multiparameter Optimization

Cruzain, the main cysteine protease of Trypanosoma cruzi, plays key roles in all stages of the parasite’s life cycle, including nutrition acquisition, differentiation, evasion of the host immune system, and invasion of host cells. Thus, inhibition of this validated target may lead to the development of novel drugs for the treatment of Chagas disease. In this study, a multiparameter optimization (MPO) approach, molecular modeling, and structure-activity relationships (SARs) were employed for the identification of new benzimidazole derivatives as potent competitive inhibitors of cruzain with trypanocidal activity and suitable pharmacokinetics. Extensive pharmacokinetic studies enabled the identification of metabolically stable and permeable compounds with high selectivity indices. CYP3A4 was found to be involved in the main metabolic pathway, and the identification of metabolic soft spots provided insights into molecular optimization. Compound 28, which showed a promising trade-off between pharmacodynamics and pharmacokinetics, caused no acute toxicity and reduced parasite burden both in vitro and in vivo.

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In vitro inhibition of Eimeria tenella sporozoite invasion into host cells by probiotics

Veterinary Parasitology ◽

10.1016/j.vetpar.2016.10.001 ◽

2016 ◽

Vol 229 ◽

pp. 93-98 ◽

Cited By ~ 10

Author(s):

S. Hessenberger ◽

G. Schatzmayr ◽

K. Teichmann

Keyword(s):

Eimeria Tenella ◽

Host Cells ◽

In Vitro Inhibition

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Prison Break: Pathogens' Strategies To Egress from Host Cells

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00024-12 ◽

2012 ◽

Vol 76 (4) ◽

pp. 707-720 ◽

Cited By ~ 54

Author(s):

Nikolas Friedrich ◽

Monica Hagedorn ◽

Dominique Soldati-Favre ◽

Thierry Soldati

Keyword(s):

Host Cell ◽

Pathogenic Bacteria ◽

Wide Spectrum ◽

Host Cells ◽

Host Immune System ◽

Intracellular Pathogens ◽

Pathogenic Yeast ◽

Apicomplexan Parasites ◽

Strategic Process ◽

Intimate Association

SUMMARYA wide spectrum of pathogenic bacteria and protozoa has adapted to an intracellular life-style, which presents several advantages, including accessibility to host cell metabolites and protection from the host immune system. Intracellular pathogens have developed strategies to enter and exit their host cells while optimizing survival and replication, progression through the life cycle, and transmission. Over the last decades, research has focused primarily on entry, while the exit process has suffered from neglect. However, pathogen exit is of fundamental importance because of its intimate association with dissemination, transmission, and inflammation. Hence, to fully understand virulence mechanisms of intracellular pathogens at cellular and systemic levels, it is essential to consider exit mechanisms to be a key step in infection. Exit from the host cell was initially viewed as a passive process, driven mainly by physical stress as a consequence of the explosive replication of the pathogen. It is now recognized as a complex, strategic process termed “egress,” which is just as well orchestrated and temporally defined as entry into the host and relies on a dynamic interplay between host and pathogen factors. This review compares egress strategies of bacteria, pathogenic yeast, and kinetoplastid and apicomplexan parasites. Emphasis is given to recent advances in the biology of egress in mycobacteria and apicomplexans.

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Thymidine uptake by chicken kidney cells parasitized by Eimeria tenella: evidence for a diffusable mediating substance

Parasitology ◽

10.1017/s0031182000056924 ◽

1981 ◽

Vol 82 (2) ◽

pp. 189-194 ◽

Cited By ~ 3

Author(s):

Catriona Urquhart

Keyword(s):

Life Cycle ◽

Dna Synthesis ◽

Host Cell ◽

Fold Increase ◽

Eimeria Tenella ◽

Thymidine Uptake ◽

Post Inoculation ◽

Kidney Cells ◽

Chicken Kidney

SUMMARYThe pattern of DNA synthesis in chicken kidney (CK) cells parasitized by Eimeria tenella was altered; a 2-fold increase in the uptake of [3H]-thymidine occurred in these cells compared with that shown by the controls. This difference was significantly different at 16 h post-inoculation (p.i.) and reached a peak at 20 h p.i. Non-parasitized cells in co-culture with parasitized cells showed a level of incorporation intermediate between that of the parasitized and non-parasitized mono- cultures. It was shown that this stimulation was due to a factor released into the medium by the parasite or the parasitized cell. A possible course of events leading to induced and prolonged DNA synthesis by the host cell and the significance of this change in the life-cycle of the parasite is discussed.

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Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella

Parasite ◽

10.1051/parasite/2018061 ◽

2018 ◽

Vol 25 ◽

pp. 60 ◽

Cited By ~ 10

Author(s):

Ming Yan ◽

Xiaoxia Cui ◽

Qiping Zhao ◽

Shunhai Zhu ◽

Bing Huang ◽

...

Keyword(s):

Protective Efficacy ◽

Body Weight Gain ◽

Expression System ◽

Protozoan Parasite ◽

Eimeria Tenella ◽

Host Cells ◽

Apicomplexan Parasites ◽

The Mean ◽

First And Second Generation ◽

Average Body

Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.

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