Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.     

The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth

The 90-kDa heat shock proteins (Hsp90) are important for stress tolerance, for newly synthesised protein folding and for the growth of various organisms. Participation of Hsp90 in the development of Apicomplexa, notably in Plasmodium falciparum and Toxoplasma gondii, has been proven. In this work, the importance of Hsp90 for Eimeria tenella, which is responsible for avian caecal coccidiosis, was studied. Our results show that E. tenella Hsp90 (EtHsp90) expression increases during infection. Immunofluorescence microscopy studies reveal a dispersed localisation of EtHsp90 during the first schizogony. Moreover, EtHsp90 is secreted by sporozoites as early as 5min after addition of FCS in a temperature-dependent manner. By using staurosporine, we invalidated the hypothesis that EtHsp90 might be a micronemal protein. Then, EtHsp90 was detected in a parasitophorous vacuole membrane. This result suggests the importance of EtHsp90 for intracellular growth of the parasite. Inhibition of EtHsp90 function using specific antibodies and geldanamicin attenuates the capacity of E. tenella to invade and grow in the host cell.     

Flotillin-1 localization on sporozoites oF Eimeria tenella

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.     

Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella o?cysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.     

Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis

Hybridoma cell lines, which secreted antibodies directed against two different strains of Eimeria tenella and one strain of E. mitis, were produced by fusion of spleen cells from sporozoite-immunized Balb/cByJ mice with P3-X63-Ag8 myeloma cells. The antibodies demonstrated at least eight different binding patterns on or in air-dried sporozoites as determined by the indirect immunofluorescent antibody (IFA) test. These patterns varied from a general internal fluorescence similar to that seen with sporozoites exposed to hyperimmune chicken serum, to fluorescence observed on the tip, pellicle, and refractile body of the parasite. Five cloned, antibody-secreting cell lines were successfully established. Four of these clones produced antibody that reacted only with various strains of E. tenella and cross-reacted with no other species of coccidia. The fifth clone produced antibody directed against only E. mitis and did not react with any other coccidial species.     

Eimeria tenella: immunogenicity of arrested sporozoites in chickens

Groups of chickens were medicated with the anticoccidial drug, decoquinate, and starting 1 day after this medication they were given daily inoculations of either 1 X 10(4) (Experiment 1) or 1 X 10(5) (Experiment 2) oocysts of a decoquinate-sensitive strain of Eimeria tenella. This assured the presence of large numbers of drug-inhibited sporozoites in the cecal tissues. The immunity arising from the presence of these inhibited sporozoites was assessed by challenging the medicated chickens with a 2.5 X 10(5) oocysts of a decoquinate-resistant strain of E. tenella. The response to challenge was assessed by weight gain, the severity of cecal lesions, hematocrits, and cecal oocyst numbers. The inhibited sporozoites promoted little (if any) immunity judged by clinical signs of disease. However, judged by body weight changes after challenge, the presence of inhibited sporozoites provided substantial protection against the body-weight-depressing effects of the challenge dose. These findings emphasize the importance of stage-specific antigen expression in Eimeria spp. infections and support the notion that immunogenicity is associated with tropic stages of the parasite.     

Establishment of Eimeria tenella (local isolate) in chicken embryos

Development of an in vitro Eimeria (E.) tenella model could be valuable as a tool for vaccine, coccidiostats or molecular biology research. 1.0 × 10,000 sporozoites per 0.1 mL were inoculated into the allantoic cavity of ten-day-old chicken embryos. The complete life-cycle of E. tenella was accomplished in eight-nine days at 37 °C and 70% humidity. The addition of 100 U insulin to the embryos could remarkably improve the output of oocysts. The development of the parasite within the embryos was systematically observed, allowing guidelines to be set regarding the appropriate times at which different developmental stages of the parasite may be sampled.     

Expression of flotillin-1 on Eimeria tenella sporozoites and its role in host cell invasion

Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells.     

Enzyme variation in Eimeria species of the chicken.

A method for the biochemical identification of protozoa belonging to the genus Eimeria is described for the first time. Starch gel electrophoresis of the enzymes lactate dehydrogenase, glucose phosphate isomerase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from parasite extracts revealed both intra- and inter-species differences when 11 strains representative of 6 species of Eimeria were examined. Oocysts were the most accessible parasite stage for investigation but sporozoites and merozoites of an embryo-adapted strain of E. tenella were also examined for enzyme activity.     

Integrating genetics and genomics to identify new leads for the control of Eimeria spp

Eimerian parasites display a biologically interesting range of phenotypic variation. In addition to a wide spectrum of drug-resistance phenotypes that are expressed similarly by many other parasites, the Eimeria spp. present some unique phenotypes. For example, unique lines of Eimeria spp. include those selected for growth in the chorioallantoic membrane of the embryonating hens egg or for faster growth (precocious development) in the mature host. The many laboratory-derived egg-adapted or precocious lines also share a phenotype of a marked attenuation of virulence, the basis of which is different as a consequence of the in ovo or in vivo selection procedures used. Of current interest is the fact that some wild-type populations of Eimeria maxima are characterized by an ability to induce protective immunity that is strain-specific. The molecular basis of phenotypes that define Eimeria spp. is now increasingly amenable to investigation, both through technical improvements in genetic linkage studies and the availability of a comprehensive genome sequence for the caecal parasite E. tenella. The most exciting phenotype in the context of vaccination and the development of new vaccines is the trait of strain-specific immunity associated with E. maxima. Recent work in this laboratory has shown that infection of two inbred lines of White Leghorn chickens with the W strain of E. maxima leads to complete protection to challenge with the homologous parasite, but to complete escape of the heterologous H strain, i.e. the W strain induces an exquisitely strain-specific protective immune response with respect to the H strain. This dichotomy of survival in the face of immune-mediated killing has been examined further and, notably, mating between a drug-resistant W strain and a drug-sensitive H strain leads to recombination between the genetic loci responsible for the specificity of protective immunity and resistance to the anticoccidial drug robenidine. Such a finding opens the way forward for genetic mapping of the loci responsible for the induction of protective immunity and integration with the genome sequencing efforts.     

The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.     

The Plasmodium falciparum heat shock protein 40, Pfj4, associates with heat shock protein 70 and shows similar heat induction and localisation patterns

Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp70-1, were both localised to the parasites nucleus and cytoplasm. In some cases, Pfj4 was also detected in the erythrocyte cytoplasm of infected erythrocytes. Immunoprecipitation studies and size exclusion chromatography indicated that Pfj4 and PfHsp70-1 may directly or indirectly interact. Our results suggest a possible involvement of Pfj4 together with PfHsp70-1 in cytoprotection, and therefore, parasite survival inside the erythrocyte.     

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to rodent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations sugest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is coregulated.     

Pancreatic chymotrypsin as the essential enzyme for excystation of Eimeria tenella.

Sporocysts from the protozoan parasite, Eimeria tenella, were isolated, preincubated with sodium taurocholate, and treated with various preparations of pancreatic enzymes. Crude trypsin, crude lipase, and purified alpha-chymotrypsin all could break the shells of sporocysts and release sporozoites. Purified trypsin was much less active than crude trypsin and purified lipase showed no activity at all. Specific inhibitors of chymotrypsin, tosyl-L-phenylalanyl chloromethane, diphenylcarbamyl chloride, and chymostatin inhibited the release of sporozoites by all the enzyme samples, whereas tosyl-L-lysyl chloromethane, a specific inhibitor of trypsin, exerted no inhibitory effect. It is thus postulated that chymotrypsin, not trypsin, is an essential enzyme involved in excystation of E. tenella. Purified chymotrypsin is recommended to replace crude trypsin in the vitro excystation of E. tenella as a likely improved procedure.     

Evolvability of Hsp70 expression under artificial election for inducible thermotolerance in independent populations of Drosophila melanogaster

To test whether expression of the inducible heat-shock protein Hsp70 increases under selection for inducible thermotolerance in Drosophila melanogaster, we performed artificial selection on replicate sets of Drosophila lines founded from two independent populations. Selection entailed pretreatment at 36 degrees C to induce thermotolerance and Hsp70 expression, followed by a more severe heat shock, whose temperature varied between sexes and among generations to achieve 50% mortality. Inducible thermotolerance increased slowly and continuously in selected lines and was 37%-50% greater than in controls after 10-11 generations. Lines founded from the two populations differed in their coevolution of Hsp70 expression. In lines founded from Evolution Canyon, Israel, Hsp70 level initially increased and thereafter was unchanged; replicate lines exhibited two temporal patterns of response to selection. In lines founded from Australia, Hsp70 levels increased throughout selection. In both cases, however, the increase in Hsp70 level averaged only 15%, suggesting that pleiotropy in Hsp70 function constrains evolutionary increase in its expression.     

The endogenous development of virulent strains and attenuated precocious lines of Eimeria tenella and E. necatrix

Studies were made of the endogenous development of the H strains of E. tenella and E. necatrix, and precocious lines derived from them. Second-generation meronts of both precocious lines matured at a faster rate than those of the parent strains. The first- and second-generation meronts of the precocious line of E. necatrix and the second-generation meronts of the precocious line of E. tenella were significantly smaller and contained fewer merozoites than the corresponding stages of the parent strains. These observations provide reasons for the precocity, and a partial explanation for the attenuation, of these lines.