The Complexity of Piroplasms Life Cycles

Although apicomplexan parasites of the group Piroplasmida represent commonly identified global risks to both animals and humans, detailed knowledge of their life cycles is surprisingly limited. Such a discrepancy results from incomplete literature reports, nomenclature disunity and recently, from large numbers of newly described species. This review intends to collate and summarize current knowledge with respect to piroplasm phylogeny. Moreover, it provides a comprehensive view of developmental events of Babesia, Theileria, and Cytauxzoon representative species, focusing on uniform consensus of three consecutive phases: (i) schizogony and merogony, asexual multiplication in blood cells of the vertebrate host; (ii) gamogony, sexual reproduction inside the tick midgut, later followed by invasion of kinetes into the tick internal tissues; and (iii) sporogony, asexual proliferation in tick salivary glands resulting in the formation of sporozoites. However, many fundamental differences in this general consensus occur and this review identifies variables that should be analyzed prior to further development of specific anti-piroplasm strategies, including the attractive targeting of life cycle stages of Babesia or Theileria tick vectors

Establishment of Babesia laboratory model and its experimental applications

Du fait de l'incidence croissante des infections à Babesia spp., Protozoaire parasite transmis par les tiques, la babésiose est considérée comme maladie émergente en médecine humaine et vétérinaire. Cette thèse apporte un aperçu de la biologie de deux agents principaux de la babésiose humaine, Babesia microti etBabesia divergens. Un modèle quantifié du parasite Babesia est proposé, permettant une étude détaillée de son cycle de vie et l'identification de molécules jouant un rôle dans son acquisition/transmission par le vecteur Ixodes ricinus. Des informations nouvelles et détaillées sur la dissémination de Babesia dans les tissus de la tique sont apportées par les techniques de visualisation/quantification développées. Le développement parasitaire dans les glandes salivaires est particulièrement étudié, car premier site responsable de la transmission du vecteur à l'hôte. Par inactivation ciblée de gènes, nous avons exploré les voies du système immunitaire de la tique, dont les molécules effectrices, et évalué leur rôle dans l'acquisition de Babesia. Une étude détaillée de l'engagement de Babesia dans la phase sexuée est réalisée, en suivant sa cinétique sous l'effet de divers stimuli. De plus, une voie nouvelle de lutte antiparasitaire est proposée envalidant le protéasome de Babesia en tant que cible thérapeutique. D'une façon générale, les travaux présentés élargissent les connaissances actuelles de la biologie du parasite Babesia incluant les interactions moléculaires à l'interface tique-Babesia et contribuent donc au contrôle de la babésiose.Growing incidence of infections caused by the ticktransmittedprotozoan parasite Babesia spp. definesbabesiosis as an emerging disease from the aspect ofhuman and veterinary medicine. The thesis provides aninsight to biology of two main agents of humanbabesiosis, Babesia microti and Babesia divergens. Weintroduce here the fully optimized quantification model ofBabesia parasite enabling the detailed investigation ofthe parasite developmental cycle and identification ofmolecules playing a role in its acquisition andtransmission by the vector Ixodes ricinus. Novel anddetailed information about Babesia dissemination withinthe tick tissues are given by newly implementedvisualization and quantification techniques. Specialemphasis is paid to parasite development in the ticksalivary glands, the primary site responsible for parasitetransmission from the vector into the host. Using genespecificsilencing we screen the tick immune pathwaysincluding effector molecules and evaluate their role inBabesia acquisition. We also provide a detailed view toBabesia parasite sexual commitment by monitoring itskinetics upon various stimuli. Moreover, a new directionof anti-babesial therapy is proposed by validation of theBabesia proteasome as a drug target. Overall, theresearch presented in the thesis extends the currentknowledge of the Babesia parasite biology includingmolecular interactions at the tick-Babesia interface andthereby could significantly contribute to a potentialcontrol of babesiosis

Babesia life cycle – When phylogeny meets biology

Although Babesia represents an important worldwide veterinary threat and an emerging risk to humans, this parasite has been poorly studied as compared to Plasmodium, its malaria-causing relative. In fact, Babesia employs highly specific survival strategies during its intraerythrocytic development and its intricate journey through the tick vector. This review introduces a substantially extended molecular phylogeny of the order Piroplasmida, challenging previous taxonomic classifications. The intriguing developmental proficiencies of Babesia are highlighted and compared with those of other haemoparasitic Apicomplexa. Molecular mechanisms associated with distinctive events in the Babesia life cycle are emphasized as potential targets for the development of Babesia-specific treatments.Instituto de PatobiologíaFil: Jalovecka, Marie. Czech Academy of Sciences. Institute of Parasitology, Biology Centre; República Checa. University of South Bohemia. Faculty of Science; República ChecaFil: Sojka, Daniel. Czech Academy of Sciences. Institute of Parasitology, Biology Centre; República ChecaFil: Ascencio, Mariano E. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The complexity of piroplasms life cycles

Although apicomplexan parasites of the group Piroplasmida represent commonly identified global risks to both animals and humans, detailed knowledge of their life cycles is surprisingly limited. Such a discrepancy results from incomplete literature reports, nomenclature disunity and recently, from large numbers of newly described species. This review intends to collate and summarize current knowledge with respect to piroplasm phylogeny. Moreover, it provides a comprehensive view of developmental events of Babesia, Theileria, and Cytauxzoon representative species, focusing on uniform consensus of three consecutive phases: (i) schizogony and merogony, asexual multiplication in blood cells of the vertebrate host; (ii) gamogony, sexual reproduction inside the tick midgut, later followed by invasion of kinetes into the tick internal tissues; and (iii) sporogony, asexual proliferation in tick salivary glands resulting in the formation of sporozoites. However, many fundamental differences in this general consensus occur and this review identifies variables that should be analyzed prior to further development of specific anti-piroplasm strategies, including the attractive targeting of life cycle stages of Babesia or Theileria tick vectors

Stimulation and quantification of Babesia divergens gametocytogenesis

Background Babesia divergens is the most common blood parasite in Europe causing babesiosis, a tick-borne malaria-like disease. Despite an increasing focus on B. divergens, especially regarding veterinary and human medicine, the sexual development of Babesia is poorly understood. Development of Babesia sexual stages in the host blood (gametocytes) plays a decisive role in parasite acquisition by the tick vector. However, the exact mechanism of gametocytogenesis is still unexplained. Methods Babesia divergens gametocytes are characterized by expression of bdccp1, bdccp2 and bdccp3 genes. Using previously described sequences of bdccp1, bdccp2 and bdccp3, we have established a quantitative real-time PCR (qRT-PCR) assay for detection and assessment of the efficiency of B. divergens gametocytes production in bovine blood. We analysed fluctuations in expression of bdccp genes during cultivation in vitro, as well as in cultures treated with different drugs and stimuli. Results We demonstrated that all B. divergens clonal lines tested, originally derived from naturally infected cows, exhibited sexual stages. Furthermore, sexual commitment was stimulated during continuous growth of the cultures, by addition of specific stress-inducing drugs or by alternating cultivation conditions. Expression of bdccp genes was greatly reduced or even lost after long-term cultivation, suggesting possible problems in the artificial infections of ticks in feeding assays in vitro. Conclusions Our research provides insight into sexual development of B. divergens and may facilitate the development of transmission models in vitro, enabling a more detailed understanding of Babesia-tick interactions

Validation of Babesia proteasome as a drug target

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis. Keywords: Proteasome, Babesia, Carfilzomib, Epoxyketone, Cytotoxicit

Additional file 3: Figure S2. of Stimulation and quantification of Babesia divergens gametocytogenesis

Optimization of qRT-PCR. Standard curves of reference and target genes (A) and qRT-PCR parameters (B). Ct = cycle threshold, R2 = correlation coefficient. (PDF 181 kb

Additional file 1: Table S1. of Stimulation and quantification of Babesia divergens gametocytogenesis

List of B. divergens strains used in this study (a – gene polymorphism analysis, b – analysis of expression of bdccp genes). (DOC 37 kb

Additional file 6: Figure S5. of Stimulation and quantification of Babesia divergens gametocytogenesis

Continuous culture growth. Relative expression of bdccp genes (A) and parasitemia levels (B) during the continuous growth of B. divergens clone 2210A G2. Gene expression was normalized using the gapdh reference gene. The expression in the highest individual replicate 1 DPI was set at 100 % and all other values were expressed relative to this. (PDF 48 kb