Experimental vaccination of chicks with Plasmodium gallinaceum sporozoites. I. Circumsporozoite proteins are expressed by sporozoites recovered from both salivary glands and midguts of mosquitoes

Immunogenicity of Plasmodium gallinaceum sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8-9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental Vaccination of Chicks with Plasmodium gallinaceum Sporozoites. I. Circumsporozoite Proteins Are Expressed by Sporozoites Recovered from Both Salivary Glands and Midguts of Mosquitoes1

Immunogenicity of Plasmodium gallinaceum Sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8–9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal. This is an advantage since obtaining the midguts is less tedious, as well as more efficient and faster.     

The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts.

The ookinete is a motile form of the malaria parasite that travels from the midgut lumen of the mosquito, invades the epithelial cells and settles beneath the basal lamina. The events surrounding cessation of ookinete motility and its transformation into an oocyst are poorly understood, but interaction between components of the basal lamina and the parasite surface has been implicated. Here we report that interactions occur between basal lamina constituents and ookinete proteins and that these interactions inhibit motility and are likely to be involved in transformation to an oocyst. Plasmodium berghei ookinetes bound weakly to microtitre plate wells coated with fibronectin and much more strongly to wells coated with laminin and collagen IV. A 1:1 mixture of collagen and laminin significantly enhanced binding. Binding increased with time of incubation up to 10 h and different components showed different binding profiles with time. Two parasite molecules were shown to act as ligands for basal lamina components. Western blots demonstrated that the surface molecule Pbs21 bound strongly to laminin but not to collagen IV whereas a 215 kDa molecule (possibly PbCTRP) bound to both laminin and collagen IV. Furthermore up to 90% inhibition of binding of ookinetes to collagen IV/laminin combination occurred if parasites were pre-incubated with anti-Pbs21 monoclonal antibody 13.1. Some transformation of ookinetes to oocysts occurred in wells coated with laminin or laminin/collagen IV combinations but collagen IV alone did not trigger transformation. No binding or transformation occurred in uncoated wells. Our data support the suggestion that ookinete proteins Pbs21 and a 215 kDa protein may have multiple roles including interactions with midgut basal lamina components that cause binding, inhibit motility and trigger transformation.     

Characterization of a developmentally regulated oocyst protein from Eimeria tenella

Changes in proteins during sporulation of Eimeria tenella oocysts were investigated. Unsporulated E. tenella oocysts collected from cecal tissue at 7 days postinoculation were sporulated in aerated media at 28 C for 0-48 hr. Gel analysis of soluble protein extracts prepared from oocysts from their respective time points indicated the presence of 2 prominent bands with relative molecular weight (Mr) in the range of 30 kDa and making up 20% of the total protein. These 2 bands, designated as major oocyst proteins (MOPs), were absent or barely detectable by 21 hr of sporulation. MOP bands were weakly reactive with glycoprotein stain but showed no mobility shift on deglycosylation. By gel analysis it was shown that the purified MOPs consisted of 2 bands of Mr 28.7 and 30.1 kDa. However, by matrix-assisted laser deabsorption-time of flight analysis it was shown that masses were about 17% lower. Internal sequence analysis of the 28.7-kDa protein generated 2 peptides of 17 and 14 amino acids in length, consistent with a recently described protein coded by the gam56 gene and expressed in E. maxima gametocytes. Rabbit antibodies made against MOPs were localized to outer portions of sporocysts before excystment and to the apical end of in vitro-derived sporozoites. These same antibodies were found to react with bands of Mr 101 and 65 kDa by Western blot but did not recognize MOPs in soluble or insoluble sporozoite extracts. The data suggest that the MOPs are derived from part of a gametocyte protein similar to that coded by gam56 and are processed during sporulation into sporocyst and sporozoite proteins. Alternatively, the binding of anti-MOP to 101- and 65-kDa proteins may result from alternatively spliced genes as the development of parasite proceeds.     

Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation

Toxoplasma gondii sporozoites possess an array of stage-specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site-specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.     

Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium

The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.     

Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host

The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).     

Sporogonic Development of Leucocytozoon smithi

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30–50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

Sporogonic development of Leucocytozoon smithi.

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30-50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.     

Identification of Plasmodium berghei Oocyst Rupture Protein 2 (ORP2) domains involved in sporozoite egress from the oocyst

Sporozoites are the infective form of malaria parasites which are transmitted from the mosquito salivary glands to a new host in a mosquito blood meal. The sporozoites develop inside the sporogonic oocyst and it is crucial for the continuation of the life cycle that the oocyst ruptures to release sporozoites. We recently described two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2), localized at the oocyst capsule, that are each essential for rupture of the oocysts. Both ORPs contain a histone fold domain implicated in the mechanism of oocyst rupture, possibly through the formation of a heterodimer between the two histone fold domains. To gain an understanding of the function of the different regions of the ORP2 protein, we generated deletion mutants. We monitored oocyst formation and rupture as well as sporozoites in the salivary gland. Our results show that different regions of ORP2 play independent roles in sporozoite egress. Deleting the N-terminal histone fold domain of ORP2 blocked sporozoite egress from the oocyst. Progressive deletions from the C-terminal resulted in no or significantly impaired sporozoite egress.     

The fine structure of the developing oocyst of Pterospora floridiensis (Apicomplexa,Urosporidae)

Developing oocysts of the gregarine Pterospora floridiensis Landers 2001 were examined by transmission electron microscopy. Each oocyst had an outer capsule and an inner capsule that contained 8 sporozoites. In early stages of development the inner capsular wall was separated from the developing sporozoites and residual mass, and was not appressed to the sporozoites. Early stage sporozoites were connected to a residual mass and were filled with endoplasmic reticulum, golgi and numerous developing secretory vesicles. In late stages of oocyst and sporozoite development, the inner capsular wall was closely appressed to the sporozoite surface. The inner capsular wall was ～60-100 nm thick and the outer capsular wall was ～160-320 nm thick. There were no extensions on the outer wall for which the genus was named. Late stage sporozoites had no residual mass connection, were more electron dense, and contained three distinct types of dense secretory structures: 1) small oval/spherical dense vesicles, 2) large (350-400 nm) vesicles near the anterior end, and 3) elongated dense tubular bodies that converged at the apex. Few ultrastructural reports exist of developing gregarine oocysts and sporozoites, and as more studies are completed these morphological characteristics may be important in interpreting molecular phylogenetic analyses.     

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.     

Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.     

A simple method of DNA extraction for Eimeria species

A new, simple method is described for extracting DNA from coccidia (Eimeriidae) oocysts. In our hands this method works well for all Eimeria oocysts and, presumably, will work equally well for oocysts of other coccidia genera. This method combines the two steps of breaking oocyst and sporocyst walls, and dissolving the sporozoite membrane in one step. This greatly simplifies the currently used DNA extraction procedures for Eimeria species and overcomes the disadvantages of existing DNA extraction methods based on glass-bead grinding and sporozoite excystation procedures. Because all the procedures are done in a 1.5-ml microfuge tube, which minimizes the loss of DNA in the extraction procedures, this method is especially suitable for samples with small number of oocysts. In addition, this method directly lyses the oocyst and sporocyst walls as well as the sporozoite membrane in a continuous incubation; therefore, it does not require the sporozoites to be alive. The results of PCR experiments indicate that this method generates better quality of DNA than what the existing glass-bead grinding method does for molecular analysis, and is suitable for both large or small number (<10(2) oocysts) of living or dead oocyst samples.     

Identification of antigens of Sarcocystis cruzi sporozoites, merozoites, and bradyzoites with monoclonal antibodies

Sporozoites and culture-derived merozoites of Sarcocystis cruzi were used to elicit monoclonal antibodies (MAb's) in mice. Some of these antibodies reacted with the surface of live sporozoites and merozoites as determined by immunofluorescence. An array of similar antigens was identified in Western blots of sporozoites by both anti-merozoite MAb's and an anti-sporozoite MAb. At least 1 antigen in blots of bradyzoites was identified by anti-merozoite MAb's and a cluster of antigens was identified by an anti-sporozoite antibody. These results indicate that several surface epitopes of sporozoites and merozoites are shared with molecules of bradyzoites and that antigen patterns of molecules bearing these epitopes in 3 stages of Sarcocystis may be either distinct or similar.     

Plasmodium berghei: relationship between protective immunity and anti-sporozoite (CSP) antibody in mice

Protective immunity and production of anti-sporozoite (CSP) antibody was studied in A/J mice injected with X-irradiated sporozoites using different immunization schedules and antigen doses. Data were also obtained on the immunogenicity of X-irradiated as compared to nonirradiated sporozoites. After a single immunization (1.5 × 10⁵ or 7.5 × 10⁴ X-irradiated sporozoites) a number of animals was completely protected when challenged, but the percentage of protected mice varied considerably from experiment to experiment. Maximal protection was obtained 7 days after the immunization. When the first injection of parasites was followed by a single booster administered 3, 4 or 5 days later, protection was considerably enhanced and the results more consistent. After a single injection of 1.5 × 10⁵ or 7.5 × 10⁴ sporozoites, CSP antibody was detectable from the 19th and 23rd day, respectively, i.e., at a time point when protection was diminishing. This antibody persisted only for a short period. When a single booster was given soon after the first injection, CSP antibody was present in the sera of all the mice from the ninth day on and persisted for greater than 80 days. A single dose of X-irradiated sporozoites injected into rats, induced antibody (CSP) formation which reached a peak after 2 weeks and persisted at this level for more than 3 months. However in rats injected with viable sporozoites, the antibody titers fell rapidly and became undetectable after 4 weeks.From these data we can conclude that (a) the immune response induced by attenuated X-irradiated sporozoites is considerably longer-lasting than that induced by viable sporozoites; (b) CSP antibodies are not detectable during the early stages of the immune response; and (c) protective immunity precedes the presence of detectable serum and antibodies.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.