The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay

ABSTRACT: Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.     

Genetic basis for GPI-anchor merozoite surface antigen polymorphism of Babesia and resulting antigenic diversity

Glycosyl-phosphatidylinositol anchor merozoite surface antigens (GPI-anchor MSA) are proposed to act in the invasion process of infective merozoites of Babesia into host erythrocytes. Because of their essential function in the survival of Babesia parasites, they constitute good candidates for the development of vaccines against babesiosis and they have been extensively analyzed. These include Babesia bovis variable MSA (VMSA) and Babesia bigemina gp45/gp55 proteins of the agents of bovine babesiosis from tropical and subtropical countries, and the Babesia divergens Bd37 and Babesia canis Bc28 proteins of the main agents of bovine and canine babesiosis in Europe, respectively. However, these are very polymorphic antigens and Babesia parasites have evolved molecular mechanisms that enable these antigens to evade the host immune system as a survival strategy. This review focuses on the genetic basis of GPI-anchor MSA polymorphism and the antigenic diversity of B-cell epitopes that might be generated in each of these Babesia species. The picture is incomplete and no Babesia genome sequence is yet available. However, the available sequences suggest that two distinct, non cross-reactive GPI-anchor MSA (i.e., with unique B-cell epitopes) may be required by all Babesia species for invasion, and that these two distinct GPI-anchor MSA would be encoded by a multigene family. Furthermore, the data are consistent with the ability of biological clones from Babesia to use these multigene families for the expression of GPI-anchor MSA, either conserved (B. canis and B. bovis) or polymorphic (B. divergens and B. bigemina) in their amino acid sequence. Moreover, as a consequence for successful parasitism, the data suggest that both conserved and polymorphic GPI-anchor MSA would present unique B-cell epitopes.     

Glycophorin A-knockout mice, which lost sialoglycoproteins from the red blood cell membrane, are resistant to lethal infection of Babesia rodhaini

Recent in vitro-based studies using several Babesia spp. have suggested that sialic acids and/or sialoglycoproteins on host red blood cells (RBCs) play an important role in their invasion of RBCs. In the present study, we analyzed the RBC characteristics of glycophorin A (GPA)-knockout mice and studied their in vivo susceptibility to lethal infection of Babesia rodhaini for the first time. In immunoblot and lectin blot analyses, glycoproteins containing O-linked oligosaccharides terminated with alpha2-3-linked sialic acids disappeared from the RBCs of GPA homozygous ((-/-)) mice. Flow cytometric analysis showed a remarkable reduction of Maackia amurensis lectin II binding to the surface of GPA(-/-) RBCs relative to control RBCs, indicating an appreciable loss of alpha2-3-linked sialic acids on the RBC surface of GPA(-/-) mice. Importantly, while B. rodhaini caused lethal infection in wild-type mice, the infected GPA(-/-) mice showed inhibition of parasite growth and eventually survived. These results indicate that RBC sialoglycoproteins lost in GPA(-/-) mice are involved in the in vivo growth of B. rodhaini, probably functioning as essential molecule(s) for the parasite invasion of host RBCs in the blood circulation.     

顶复门原虫入侵相关因子的研究进展

顶复门原虫是一类专一性的细胞内寄生原虫,包括弓形虫(Toxoplasma gondii)、隐孢子虫(Cryptosporidium spp.)、疟原虫(Plasmodium spp.)、巴贝斯虫(Babesia spp.)及艾美耳球虫(Eimeria spp.)等,是人和动物的重要病原.这类原虫具有相似的亚细胞结构和保守的入侵机制.研究结果表明,入侵过程是由大量的入侵相关蛋白分子所介导的,包括微线、棒状体及致密颗粒所分泌的相关蛋白等.随着生物信息学及分子生物学的发展,顶复门原虫入侵相关蛋白分子的研究资料也日益增多.笔者结合最近几年本课题组的研究成果,综述了顶复门原虫入侵相关蛋白因子的最新进展.     

An electron microscopic study of intra-erythrocytic stages of Babesia bovis in the brain capillaries of infected splenectomized calves.

Splenectomized vaccine donor calves undergoing primary reactions to Babesia bovis infections may develop cerebral babesiosis which leads to death if not treated in time. A brain biopsy was performed on an artificially-infected animal showing nervous symptoms and the tissue was immediately processed for electron microscopic examination. Virtually every erythrocyte in the brain capillaries sectioned was infected with B. bovis. Intra-erythrocytic merozoites, trophozoites and dividing trophozoites were indentified. Important features of the piriform merozoites included a reduced apical complex consisting of the anterior polar ring, microtubules, rhoptries and micronemes. Unidentified membrane-bound bodies, mostly spherical in shape, were observed anterior to the nucleus. The trophozoites showed very little structural differentiation and no food vacuoles or micropores could be detected. Each trophozoite produced 2 identical merozoites and the parent cell became totally incorporated in the daughter merozoites in the multiplication process. Projections were seen radiating from the surface of infected erythrocytes which appeared to adhere to other surfaces on contact. This probably resulted in the sludging of infected erythrocytes in the capillaries. The latter observations coincide with those described for Babesia argentina.     

鸡球虫入侵相关分子的研究进展

鸡球虫病是由艾美耳球虫寄生于肠道所引起的一种危害极其严重的寄生虫病,给养鸡业造成巨大的经济损失.艾美耳球虫属于顶复器门原虫,在入侵宿主细胞过程中需要通过入侵虫体顶端的顶复器分泌蛋白发挥作用.目前已报道与鸡球虫入侵相关的重要蛋白,包括微线蛋白、蛋白激酶、热激蛋白以及糖酵解酶等.这些蛋白主要参与了鸡球虫入侵宿主细胞以及在宿主细胞内的生长发育、参与了虫体的细胞周期活动以及参与了糖酵解提供虫体入侵需要的能量等,进一步对这些分子进行研究,对了解鸡球虫入侵宿主细胞的相关机理及发展抗球虫病疫苗和治疗药物将有积极的意义.     

Babesia--a historical overview

The history of the genus Babesia is briefly outlined. The classical differences with the main other genus of non-pigment-forming hemoparasites, Theileria, are the absence of extra-erythrocytic multiplication (schizogony) in Babesia and the cycle in the vector tick, which includes transovarial transmission in Babesia but only transstadial transmission in Theileria. Also, the multiplication in the red cell of Babesia, by budding, most often results in two daughter cells (merozoites), while that of Theileria gives four merozoites, often as a Maltese cross. In particular this means that what is still commonly called Babesia microti is not a Babesia and that it would be just as logical to speak of human theileriosis as of babesiosis. The small piroplasm of horses, long known as Babesia equi, is already commonly designated as Theileria equi. However, on molecular grounds, it may be necessary to create a new genus for these parasites. The Babesia species of domestic animals are briefly discussed and presented in a table.     

Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. A review

Staphylococcus aureus is the most important and prevalent contagious mammary pathogen; it causes clinical and subclinical intramammary infection with serious economic loss and herd management problems in dairy cows. In vitro studies have shown that Staphylococcus aureus adheres to mammary epithelial cells and extracellular matrix components and invades into mammary epithelial as well as other mammary cells. Staphylococcus aureus strains from intramammary infection produce several cell surface-associated and extracellular secretory products. The exact pathogenic roles of most of the products and their effects on adhesion and invasion are not well evaluated. It is also known that mammary epithelial cell-associated molecules and extracellular matrix components interact with S. aureus during the pathogenesis of mastitis, but their roles on adhesion and invasion have not been characterized. The adhesion of S. aureus to epithelial cells may involve non-specific physicochemical interactions and/or specific interactions between bacterial cell-associated ligands and host cell surface receptors. In vitro adhesion depends on the S. aureus strain, the growth phase of the bacteria, the growth medium and the origin of the epithelial cells. Adhesion is hypothesized to be a prerequisite and crucial early step for mammary gland infection. Staphylococcus aureus invades mammary epithelial cells. It also invades other cells such as endothelial cells and fibroblasts. Bacteria are found enclosed in membrane bound vacuoles in the cytoplasm of mammary epithelial cells. Recent observations indicate that S. aureus escapes from the phagosome into the cytoplasm and induces apoptosis. The invasion into mammary epithelial cells may occur through an endocytic process that requires involvement of elements of the cytoskeleton or by direct binding of bacteria to epithelial cells through a process mediated by specific receptors that needs de novo protein synthesis by both cells. Thus, the recurrent subclinical infection may result from this intracellular existence of bacteria that are protected from host defenses and effects of antibiotics. This review emphasizes on recent findings on S. aureus adhesion to mammary epithelial cells and extracellular matrix components and invasion into mammary epithelial cells.     

顶复门原虫棒状体蛋白疫苗候选分子的研究进展

顶复门原虫是一类专一性的胞内寄生性原虫,其种类丰富、分布广泛,包括艾美耳球虫、巴贝斯虫、隐孢子虫、疟原虫和弓形虫等.此类原虫具有保守的棒状体、致密颗粒以及微线等顶端复合器结构,这些细胞器可分泌大量的入侵相关蛋白分子介导虫体入侵宿主细胞.而棒状体蛋白是这类原虫在入侵过程中由棒状体分泌的一类蛋白,其在虫体入侵宿主细胞、纳虫...     

Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites

An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.     

Involvement of a host erythrocyte sialic acid content in Babesia bovis infection

Host sialic acid (SA) has recently been suggested to play an important role in erythrocyte (RBC) infection by Babesia spp. The present study attempted to further determine the specific type of SAs important in the RBC invasion. Bovine RBC was found to bear abundant alpha2-3-linked SA residues but not alpha2-6-linked SA in nature, confirmed by flow cytometric analysis of the neuraminidase (Nm)-treated RBCs. Lectin-blot analyses revealed the removal of alpha2-3-linked SAs from the 97-, 33-, and 31-kDa bands by the Nm treatment. Addition of the Nm-treated RBCs into an in vitro culture of B. bovis resulted in a decreased population of the parasitized RBCs. The thin smear samples from the cultures were then observed under a confocal laser scanning microscope after staining with the alpha2-3-linked SA-specific lectin: a selective invasion of B. bovis was found only in the intact RBCs bearing the SAs, but not in the desialylated RBCs. Furthermore, a significant reduction of the parasitized RBCs was also observed in the culture supplemented with exogenous 3'-sialyllactose containing the alpha2-3-linked SAs. However, the complete inhibition of parasite proliferation was not achieved in the culture. These findings indicate that while the alpha2-3-linked SA-dependent pathway is needed for highly efficient invasion of host RBCs by B. bovis, there might also be other potential alternative pathways.     

Continuous in vitro cultivation of a recently identified Babesia that infects small ruminants in China

Babesia sp. Xinjiang was isolated from a splenectomised sheep infested by Rhipicephalus sanguineus and Hylomma anatolicum anatolicum, collected from sheep and cattle in Xinjiang province. It was considered to be a novel ovine Babesia species on the basis of its morphology, pathogenicity, vector tick species and alignments of 18S ribosomal RNA (18S rRNA) and internal transcribed spacers (ITS) gene sequences. Continuous in vitro cultures of the ovine parasite were established using infected sheep blood. In RPMI 1640 medium with 7.5% sheep red blood cells (RBCs) maintained in an incubator at 37 °C and 5% CO(2), the percentage of parasitized erythrocytes (PPE) peaked at 10% in 24- and 6-well plates. It increased to 20-50% with the same culture medium but with 2.5% RBC in 75 cm(2) flasks. Two clonal lines of Babesia sp. Xinjiang were screened using the limiting dilution method. Growth characteristics of these lines in vitro were measured by a microtiter-based spectrophotometric method and from the PPE. The generation time in sheep erythrocytes was between 15.20 h and 16.27 h. Furthermore, the host range of parasite was identified with in vitro culture and in vivo infection. Erythrocytes of sheep, cattle, sika deer and humans could be invaded into by lines in vitro, but the parasites could not propagate in human erythrocytes. The parasites could not enter erythrocytes from goats in vitro. However, in vivo, only sheep could be infected by lines. Finally, a Babesia sp. Xinjiang-like parasite (which shared 99.5% identity with the original strain of Babesia sp. Xinjiang) was isolated using this in vitro culture system from 1 of 19 sheep blood samples collected from western Gansu province, China.     

Detection of anti-Babesia gibsoni heat shock protein 70 antibody and anti-canine heat shock protein 70 antibody in sera from Babesia gibsoni-infected dogs

Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody.     

Dermatitis in a dog associated with an unidentified Toxoplasma gondii-like parasite

Protozoal dermatitis was diagnosed in a 6-year-old female Great Dane dog from Rio de Janeiro, Brazil. The dog died because of a chronic illness with an Ehrlichia-like organism. Numerous apicomplexan parasites were identified histologically in the section of dermal lesions. The protozoan reacted with Toxoplasma gondii polyclonal rabbit serum but not with Neospora caninum or Sarcocystis neurona antibodies. Ultrastructurally, the protozoa was not T. gondii because it had schizont-like structures with merozoites arranged around a prominent residual body, and the merozoites had several rhoptries with electron-dense contents; rhoptries in T. gondii tachyzoites are electron-lucent and a residual body is not found in groups of tachyzoites. This is the first report of unidentified T. gondii-like protozoa in the skin of a dog.     

Protein analysis of Babesia caballi merozoites by two-dimensional polyacrylamide gel electrophoresis and western blotting

Babesia caballi merozoites were prepared by combining two improved methods of cultivation and purification of merozoites using Percoll-gradiation, and the protein compositions of merozoites were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The relative molecular masses of the major proteins and protein masses separated by electrophoresis were >94, 80-70, 50-45, 34-30, 30-28 and 18 kDa. By Western blotting, twelve proteins or protein groups were recognized by pooled sera from two horses experimentally infected with B. caballi. Among twelve proteins, five new proteins (54, 30-26, 24, and two 18 kDa) were identified, and the 48 kDa protein was revealed to consist of 2 components in the B. caballi merozoite. One protein (54 kDa) of B. caballi was also recognized by the pooled sera from two horses experimentally infected with B. equi.     

Advances and prospects for subunit vaccines against protozoa of veterinary importance.

Protozoa are responsible for considerable morbidity and mortality in domestic and companion animals. Preventing infection may involve deliberate exposure to virulent or attenuated parasites so that immunity to natural infection is established early in life. This is the basis for vaccines against theilerosis and avian coccidiosis. Vaccination may not be effective or practical with diseases, such as cryptosporidiosis, that primarily afflict the immune-compromised or individuals with an incompletely developed immune system. Strategies for combating these diseases often rely on passive immunotherapy using serum or colostrums containing antibodies to parasite surface proteins. Subunit vaccines offer an attractive alternative to virulent or attenuated parasites for several reasons. These include the use of bacteria or lower eukaryotes to produce recombinant proteins in batch culture, the relative stability of recombinant proteins compared to live parasites, and the flexibility to incorporate only those antigens that elicit "protective" immune responses. Although subunit vaccines offer many theoretical advantages, our lack of understanding of immune mechanisms to primary and secondary infection and the capacity of many protozoa to evade host immunity remain obstacles to developing effective vaccines. This review examines the progress made on developing recombinant proteins of Eimeria, Giardia, Cryptosporidium, Toxoplasma, Neospora, Trypanosoma, Babesia, and Theileria and attempts to use these antigens for vaccinating animals against the associated diseases.     

Effects of hybridoma antibodies on invasion of cultured cells by sporozoites of Eimeria

Hybridoma antibodies (Hab) produced against sporozoites or merozoites of four species of Eimeria were tested for the ability to inhibit the invasion of cultured primary avian kidney cells by sporozoites of Eimeria. Five of 16 Hab that were tested showed inhibitory activity. All five of these Hab were produced against sporozoites and reacted with sporozoite surface antigens or surface/internal antigens. Four Hab produced against merozoites of E. acervulina cross-reacted with sporozoite surface antigens but failed to inhibit invasion. Similarly, Hab reacting with sporozoite anterior tips or refractile bodies had little effect on invasion. Collectively, the data suggest that surface antigens or surface/internal antigens that are unique to the sporozoite stage may influence or be part of the invasion process. Indirect immunofluorescent-antibody tests and ferritin (Fe) labeling combined with electron microscopy indicated differences in binding of two of the Hab to the sporozoite surface membranes. For example, after exposure to Hab 43A6 and a fluorescein-antimouse IgG conjugate, extracellular sporozoites of E. meleagrimitis fluoresced brightly but intracellular sporozoites exhibited little fluorescent label. Sporozoites labeled with Hab 43A6 plus a ferritin-antimouse IgG conjugate that were observed in the process of cell invasion had ferritin on the extracellular portion of the parasite but not on the intracellular portion. Extracellular aggregates of ferritin were observed near the site of invasion. The data suggested that antigens of the sporozoite surface that are recognized by Hab 43A6 are "scraped off" during the invasion of cells. In contrast, after exposure to Hab E5, both extracellular and intracellular sporozoites of E. tenella fluoresced. However, ferritin label was not observed on viable sporozoites, even when they were fixed immediately after the labeling procedure. The antigens recognized by Hab E5 may be associated with parasite secretory products rather than with an integral part of the sporozoite surface membrane.     

Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.     

Characterization of stages of Hepatozoon americanum and of parasitized canine host cells

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in "onion skin" cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a "tail" in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.