Number Of Sporozoites Research Articles

Determinants of relapse periodicity in Plasmodium vivax malaria.

Plasmodium vivax is a major cause of febrile illness in endemic areas of Asia, Central and South America, and the horn of Africa. Plasmodium vivax infections are characterized by relapses of malaria arising from persistent liver stages of the parasite (hypnozoites) which can be prevented only by 8-aminoquinoline anti-malarials. Tropical P. vivax relapses at three week intervals if rapidly eliminated anti-malarials are given for treatment, whereas in temperate regions and parts of the sub-tropics P. vivax infections are characterized either by a long incubation or a long-latency period between illness and relapse - in both cases approximating 8-10 months. The epidemiology of the different relapse phenotypes has not been defined adequately despite obvious relevance to malaria control and elimination. The number of sporozoites inoculated by the anopheline mosquito is an important determinant of both the timing and the number of relapses. The intervals between relapses display a remarkable periodicity which has not been explained. Evidence is presented that the proportion of patients who have successive relapses is relatively constant and that the factor which activates hypnozoites and leads to regular interval relapse in vivax malaria is the systemic febrile illness itself. It is proposed that in endemic areas a large proportion of the population harbours latent hypnozoites which can be activated by a systemic illness such as vivax or falciparum malaria. This explains the high rates of vivax following falciparum malaria, the high proportion of heterologous genotypes in relapses, the higher rates of relapse in people living in endemic areas compared with artificial infection studies, and, by facilitating recombination between different genotypes, contributes to P. vivax genetic diversity particularly in low transmission settings. Long-latency P. vivax phenotypes may be more widespread and more prevalent than currently thought. These observations have important implications for the assessment of radical treatment efficacy and for malaria control and elimination.

Arrested Oocyst Maturation in Plasmodium Parasites Lacking Type II NADH:Ubiquinone Dehydrogenase

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.

The intradermal route for inoculation of sporozoites of rodent malaria parasites for immunological studies

Rodent malaria parasites are commonly used for investigations into the immunology of pre-erythrocytic stage malaria infection, as sporozoites can be easily produced in the laboratory. In the majority of past immunological studies using this system, sporozoites are inoculated into mice via the intravenous (IV) route. In natural situations, however, sporozoites are deposited into the skin by the bite of Anopheline mosquitoes, and it is likely that the immunological response to such natural intradermal (ID) inoculation will be different to that achieved through the IV route. Although infected mosquito bites are sometimes used during experimental induction of immunity in mice, this method is problematic because of the low numbers of sporozoites introduced to the skin and the large variation in sporozoite inoculation between individual mosquitoes. Here, we show that ID inoculation of dissected mosquito salivary gland sporozoites of Plasmodium yoelii allows the accurate introduction of known numbers of sporozoites into the skin and that these parasites successfully invade the liver. Furthermore, immunization of mice using ID inoculations of live sporozoites contemporaneously with mefloquine treatment induces an immune response that is protective against the development of liver stage parasites, and this protection does not differ significantly from that achieved with IV immunizations performed in the same manner.

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria–epithelial cell attachment, 10 5 E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO 2 incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater ( P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.

Neither Mosquito Saliva nor Immunity to Saliva Has a Detectable Effect on the Infectivity ofPlasmodiumSporozoites Injected into Mice

Malaria infection is initiated when a female Anopheles mosquito probing for blood injects saliva, together with sporozoites, into the skin of its mammalian host. Prior studies had suggested that saliva may enhance sporozoite infectivity. Using rodent malaria models (Plasmodium berghei and P. yoelii), we were unable to show that saliva had any detectable effect on sporozoite infectivity. This is encouraging for plans to immunize humans with washed, attenuated P. falciparum sporozoites because many individuals develop cutaneous, hypersensitivity reactions to mosquito saliva after repeated exposure. If washed sporozoites have no appreciable loss of infectivity, they likely do not have decreased immunogenicity; thus, vaccinees are unlikely to develop cutaneous reactions against mosquito saliva during attempted immunization with such sporozoites. Earlier studies also suggested that repeated prior exposure to mosquito saliva reduces infectivity of sporozoites injected by mosquitoes into sensitized hosts. However, our own studies show that prior exposure of mice to saliva had no detectable effect on numbers of sporozoites delivered by infected mosquitoes, the rate of disappearance of these sporozoites from the skin or infectivity of the sporozoites. Under natural conditions, sporozoites are delivered both to individuals who may exhibit cutaneous hypersensitivity to mosquito bite and to others who may have not yet developed such reactivity. It was tempting to hypothesize that differences in responsiveness to mosquito bite by different individuals might modulate the infectivity of sporozoites delivered into a milieu of changes induced by cutaneous hypersensitivity. Our results with rodent malaria models, however, were unable to support such a hypothesis.

Effect of the Probiotic Lactobacillus Plantalum CMU-FP002 on Oocyst Shedding by Broilers Inoculated with Eimeria Tenella

Coccidiosis is an infectious disease that causes the most widespread health problems in the broiler industry. This study indicated that chickens fed with the probiotic Lactobacillus plantalum CMU- FP002 exhibited an 85.63% reduction in the number of oocysts of Eimeria tenella shed in the faeces compared with the control group. The average number of oocysts shed by the group fed the probiotic and a group fed an antibiotic were both significantly (P&lt;0.05) less than the average number of oocysts shed by the control group. Coccidiosis is an infectious disease caused by a protozoan parasite of the genus Eimeria and is the most consistently reported health problem in poultry (Biggs, 19–82; Rose et al, 1987; Williams, 1999). Generally, the number of oocysts shed in the faeces is dependent on the number of sporozoites and merozoites that penetrate the enterocytes for a given inoculum dose, and so represents the infection-resistant ability of broilers. Susceptibility to Eimeria can also be assessed on the basis of the number of oocysts obtained from droppings collected for four days starting on day 6 post-inoculation (Dalloul et al., 2005).

Kinetics of Mosquito-Injected Plasmodium Sporozoites in Mice: Fewer Sporozoites Are Injected into Sporozoite-Immunized Mice

Malaria is initiated when the mosquito introduces sporozoites into the skin of a mammalian host. To successfully continue the infection, sporozoites must invade blood vessels in the dermis and be transported to the liver. A significant number of sporozoites, however, may enter lymphatic vessels in the skin or remain in the skin long after the mosquito bite. We have used fluorescence microscopy of Plasmodium berghei sporozoites expressing a fluorescent protein to evaluate the kinetics of sporozoite disappearance from the skin. Sporozoites injected into immunized mice were rapidly immobilized, did not appear to invade dermal blood vessels and became morphologically degraded within several hours. Strikingly, mosquitoes introduced significantly fewer sporozoites into immunized than into non-immunized mice, presumably by formation of an immune complex between soluble sporozoite antigens in the mosquito saliva and homologous host antibodies at the proboscis tip. These results indicate that protective antibodies directed against sporozoites may function both by reducing the numbers of sporozoites injected into immunized hosts and by inhibiting the movement of injected sporozoites into dermal blood vessels.

The temporal dynamics of Plasmodium density through the sporogonic cycle within Anopheles mosquitoes

The impact of parasite density on malaria transmission remains unclear. We investigated sporogony temporal dynamics and the effect of parasite density on these dynamics. A series of experiments was conducted in which cages of mosquitoes were fed on blood containing a range of ookinete densities. Samples of surviving mosquitoes were dissected over time post-feeding to count oocyst and sporozoite numbers. Results reveal a humped (convex) pattern of oocyst numbers and suggest that transition rates between sporogony stages are density dependent. This has implications for the design of parasite-mosquito interface studies and the development of transmission-blocking strategies.

The susceptibility of Anopheles lesteri to infection with Korean strain of Plasmodium vivax

BackgroundFollowing its recent re-emergence, malaria has gained renewed attention as a serious infectious disease in Korea. Three species of the Hyrcanusgroup, Anopheles lesteri, Anopheles sinensis and Anopheles pullus, have long been suspected malaria vectors. However, opinions about their vector ability are controversial. The present study was designed with the aim of determining the susceptibility of these mosquitoes to a Korean isolate of Plasmodium vivax. Also, An. sinensis is primarily suspected to be vector of malaria in Korea, but in Thailand, the same species is described to have less medical importance. Therefore, comparative susceptibility of Thai and Korean strains of An. sinensis with Thai strain of P. vivax may be helpful to understand whether these geographically different strains exhibit differences in their susceptibility or not.MethodsThe comparative susceptibility of An. lesteri, An. sinensis and An. pullus was studied by feeding laboratory-reared mosquitoes on blood from patients carrying gametocytes from Korea and Thailand.ResultsIn experimental feeding with Korean strain of P. vivax, oocysts developed in An. lesteri, An. sinensis and An. pullus. Salivary gland sporozoites were detected only in An. lesteri and An. sinensis but not in An. pullus. Large differences were found in the number of sporozoites in the salivary glands, with An. lesteri carrying much higher densities, up to 2,105 sporozoites in a single microscope field of 750 × 560 μM, whereas a maximum of 14 sporozoites were found in any individual salivary gland of An. sinensis. Similar results were obtained from a susceptibility test of two different strains of An. sinensis to Thai isolate of P. vivax, and differences in vector susceptibility according to geographical variation were not detected.ConclusionThe high sporozoite rate and sporozoite loads of An. lesteri indicate that this species is highly susceptible to infection with P. vivax. Anopheles sinensis appears to have a markedly reduced ability to develop salivary gland infection, whilst in An. pullus, no sporozoites were found in the salivary glands. Provided that the survival rate of An. lesteri is sufficiently high in the field, it would be a highly competent vector of vivax malaria.

Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14–15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10–20% and 3–10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.

Improved excystation protocol for Eimeria nieschulzi (Apikomplexa, Coccidia)

Large numbers of sporozoites are a crucial prerequisite for in vitro experiments with Eimeria species. There are no protocols to obtain high amounts of vital purified sporozoites of Eimeria nieschulzi; therefore, an improved excystation protocol is urgently needed. Most excystation procedures for Eimeria oocysts use a mechanical disruption method for the release of sporocysts, assuming that oocyst disruption of Eimeria does not require enzymes (proteases). However, rodent Eimeria oocysts are susceptible to pepsin digestion (Kowalik S, Zahner H (1999) Eimeria separata: method for the excystation of sporozoites. Parasitol Res 85:496-499). Here, we describe a method that combines enzymatic treatment of oocyst walls before the mechanical disruption with glass beads. Using this protocol, we achieved an up to fivefold increase of free viable sporozoites of E. nieschulzi and could significantly shorten the time of excystation. These results confirm the assumption that rodent Eimeria species, in contrast to Eimeria species of birds, possess protease sensitive oocysts.

Identification and molecular characterization of a novel protein Saglin as a target of monoclonal antibodies affecting salivary gland infectivity of Plasmodium sporozoites.

Molecular mechanisms underlying the interaction between malarial sporozoites and putative receptor(s) on the salivary glands of Anopheles gambiae remain largely unknown. In previous studies, a salivary gland protein of ~100 kDa was identified as a putative target based on recognition of the protein by a monoclonal antibody (mAb) 2A3 that caused a >/= 70% reduction in the average number of sporozoites per infected salivary gland when fed to mosquitoes. Using affinity purification we purified the target of this mAb from extracts of female A. gambiae salivary glands and it was found to be a novel protein by tandem mass spectrometric analysis. Biochemical and molecular characterization of the 100 kDa protein showed that this molecule, designated Saglin, exists as a disulphide-bonded homodimer of 50 kDa subunits. The ability to form homodimers was retained even in the recombinant Saglin expressed in mammalian cells (HEK293). The amino acid sequence of Saglin contains a signal peptide suggesting that Saglin is a secreted protein. If Saglin is indeed involved in the process of invasion of A. gambiae salivary glands by sporozoites of Plasmodium, it could provide a novel target for future investigations aimed at interruption of malaria transmission.

Direct Microscopic Quantification of Dynamics of Plasmodium berghei Sporozoite Transmission from Mosquitoes to Mice

The number of malaria sporozoites delivered to a host by mosquitoes is thought to have a significant influence on the subsequent course of the infection in the mammalian host. We did studies with Anopheles stephensi mosquitoes with salivary gland infections of Plasmodium berghei sporozoites expressing a red fluorescent protein. After individual mosquitoes fed on an ear pinna or the ventral abdomen of a mouse, fluorescence microscopy was used to count numbers of sporozoites. Mosquitoes allowed to feed on the ear for periods of 3 versus 15 min deposited means of 281 versus 452 sporozoites, respectively, into the skin; this may have epidemiological implications because mosquitoes can feed for longer periods of time on sleeping hosts. Mosquitoes feeding on the ventral abdomen injected sporozoites not only into the skin but also into the underlying peritoneal musculature. Although mosquitoes injected fewer sporozoites into the abdominal tissues, more of these were reingested into the mosquito midgut, probably a consequence of easier access to blood intake from the abdominal area. The most consistent parameter of sporozoite transmission dynamics under all conditions of mosquito probing and feeding was the relatively slow release rate of sporozoites (approximately 1 to 2.5 per second) from the mosquito proboscis. The numbers of sporozoites introduced into the host by mosquitoes and the transmission efficiencies of sporozoite delivery are multifactorial phenomena that vary with length of probing time, skin site being fed upon, and numbers of sporozoites within the salivary glands.

Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel

For successful transmission to the vertebrate host, malaria sporozoites must migrate from the mosquito midgut to the salivary glands. Here, using purified sporozoites inoculated into the mosquito haemocoel, we show that salivary gland invasion is inefficient and that sporozoites have a narrow window of opportunity for salivary gland invasion. Only 19% of sporozoites invade the salivary glands, all invasion occurs within 8h at a rate of approximately 200 sporozoites per hour, and sporozoites that fail to invade within this time rapidly die and are degraded. Then, using natural release of sporozoites from oocysts, we show that haemolymph flow through the dorsal vessel facilitates proper invasion. Most mosquitoes had low steady-state numbers of circulating sporozoites, which is remarkable given the thousands of sporozoites released per oocyst, and suggests that sporozoite degradation is a rapid immune process most efficient in regions of high haemolymph flow. Only 2% of Anopheles gambiae haemocytes phagocytized Plasmodium berghei sporozoites, a rate insufficient to explain the extent of sporozoite clearance. Greater than 95% of haemocytes phagocytized Escherichia coli or latex particles, indicating that their failure to sequester large numbers of sporozoites is not due to an inability to engage in phagocytosis. These results reveal the operation of an efficient sporozoite-killing and degradation machinery within the mosquito haemocoel, which drastically limits the numbers of infective sporozoites in the mosquito salivary glands.

RE-INGESTION OF PLASMODIUM BERGHEI SPOROZOITES AFTER DELIVERY INTO THE HOST BY MOSQUITOES

Malaria-infected mosquitoes feeding on a mammalian host inject sporozoites into the skin to induce a malaria infection. The numbers of sporozoites ultimately able to reach the liver may be important determinants of the characteristics of the ensuing blood infection. Because feeding mosquitoes not only inject sporozoites into the host but concomitantly ingest blood to obtain their bloodmeal, some sporozoites are re-ingested by the feeding mosquito. We studied transmission of fluorescent Plasmodium berghei sporozoites injected into mice by Anopheles stephensi mosquitoes and found that the numbers of sporozoites re-ingested by mosquitoes are comparable to numbers previously reported to be delivered directly into mice. Thus, re-ingestion of sporozoites likely plays a significant role in transmission dynamics of malaria by mosquitoes, and may account for the failure of some sporozoite-infected mosquitoes to induce a blood infection.

Invasion of the intestinal tract by sporozoites of Eimeria coecicola and Eimeria intestinalis in naive and immune rabbits

Naive and immune specific-pathogen-free rabbits were inoculated in the duodenum with sporocysts of Eimeria coecicola or Eimeria intestinalis. Samples were taken from the following tissues: duodenum (site of penetration of sporozoites), ileum (specific target site of the endogenous development of E. intestinalis), vermiform appendix (target site of E. coecicola) and two extraintestinal sites, mesenteric lymph nodes (MLNs), and spleen. The presence of sporozoites was checked by immunohistochemistry. In rabbits primary-infected with E. coecicola, large numbers of sporozoites were detected in the duodenum, extraintestinal sites, and vermiform appendix. The abundance of sporozoites in the spleen, MLN, and appendix was significantly reduced in the immune rabbits, and the migration seemed impeded. In the rabbits infected with E. intestinalis, sporozoites were absent in the spleen and MLN, indicating that the route of migration is different from that of E. coecicola. The number of sporozoites in the crypts of the ileum was markedly reduced in the immune animals.

Exit of Plasmodium Sporozoites from Oocysts Is an Active Process That Involves the Circumsporozoite Protein

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.

Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes.

Malaria transmission begins with the injection of Plasmodium sporozoites into the skin of a vertebrate host by infected anopheline mosquitoes. Although the size of the sporozoite inoculum likely affects the course of the disease, the number of sporozoites injected by infected mosquitoes has not been determined in vivo. Using a quantitative PCR assay, we determined the number of sporozoites injected into mice by single mosquitoes. Analysis of 59 mosquito feedings showed that a single infected mosquito injected between 0 and 1,297 sporozoites, with a mean of 123 and a median of 18. Twenty-two percent of infected mosquitoes injected no sporozoites. The number of sporozoites injected was only weakly correlated to the salivary gland load. To better understand the large variability in sporozoite injection among mosquitoes, we quantified the sporozoites injected by individual mosquitoes on three different days. Approximately 20% of moderately to heavily infected mosquitoes injected few to no sporozoites on all 3 days, suggesting that some mosquitoes are poor transmitters of sporozoites. Other mosquitoes injected high numbers of sporozoites on at least one of the days observed and minimal numbers on the other day(s), supporting the hypothesis that sporozoite injection is discontinuous, a pattern that may aid in the establishment of malaria infection.

COMPARATIVE SUSCEPTIBILITY OF THREE SPECIES OF ANOPHELES FROM BELIZE, CENTRAL AMERICA, TO PLASMODIUM FALCIPARUM (NF-54)

In August of 2000, a comparative susceptibility study was conducted using 3 species of Anopheles mosquitoes from Belize, Central America, and a standard species used in laboratory infection studies, Anopheles stephensi. Test populations were fed human blood infected with cultured Plasmodium falciparum (NF-54 strain) parasites via a membrane feeder. The control species, An. stephensi, exhibited the highest infections, with 73.8% of dissected specimens positive for sporozoites in the salivary glands. The control species also showed heavier sporozoite loads; 74.0% of positive glands having greater than 200 sporozoites. Of species from Belize, Anopheles darlingi was the most susceptibile, e.g., 41.0% of salivary glands were positive, with more than 200 sporozoites per gland. Anopheles vestitipennis had a low salivary gland infection rate (9.3%) and a moderate number of sporozoites in glands (i.e., 85.7% containing 50-250 sporozoites). Anopheles albimanus was the least susceptible species to infection. No specimens of An. albimanus from the Golden Stream population developed sporozoites in the salivary glands, yet 20.7% of dissected specimens had positive midgut infections. The An. albimanus Buena Vista population showed similar results with only a 2.2% salivary gland infection rate and a 21.5% midgut infection rate. Oocysts in An. stephensi increased in size by 20% after day 10. Development peaked at day 12, with a mean oocyst diameter of 58 microm at onset of oocyst differentiation. Oocysts developed more slowly in An. vestitipennis until day 10. After day 10, there was a 53% increase in oocyst development over the previous 10 days. Oocyst differentiation was not observed until day 13 postfeed. As with An. vestitipennis, both populations of An. albimanus showed similar slow rates of oocyst development; however, no dramatic growth increase occurred after day 10. The oocysts in the Golden Stream population exhibited a cessation of growth after day 10, peaking at a mean of 30 microm. The Buena Vista population did not exhibit the same level of reduced oocyst development. A gradual increase in growth continued until days 13 and 14 (36.7 and 35.7 microm, respectively).

Babesia bigemina: Sporozoite Isolation from Boophilus microplus Nymphs and Initial Immunomolecular Characterization

It has been hypothesized that babesial sporozoites express specific antigens that induce protective immunologic responses in cattle. However, they remain uncharacterized, partly for lack of research on the sporozoite stage of Babesia spp. This field suffers from complete knowledge of parasite development in the tick salivary gland; limited amounts of sporozoites from ticks, and a lack of protocols for induction and purification of sporozoites. In this work, Boophilus microplus larvae infected with B. bigemina were fed on susceptible cattle. Nymphs were collected and macerates were separated by a Percoll density gradient. Microscopic analysis of Giemsa-stained smears showed a larger number of sporozoites from nymphs fed for 9 days. Percoll-purified sporozoites were observed in large numbers in groups or individually and free of tick cells. RT-PCR analysis of total RNA extracted from purified sporozoites indicated transcription of the rhoptry associate protein 1 (rap-1) genes: rap-1a, rap-b, rap-1c, as well as the heat shock protein 20 (hsp-20) gene. Purified sporozoites were cultured in vitro analyzed for RAP-1a expression using an immunocytochemistry assay. Erythrocyte-attached sporozoites reacted with a specific RAP-1a monoclonal antibody. This is the first report of Babesia bigemina sporozoite antigens. Moreover, purified sporozoites will allow the characterization of stage-specific antigens involved in immunologic protection.