Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants.

Soluble parasite antigens (SPA) from different Babesia species have been shown earlier to induce protective immunity when used as vaccine. However, initial attempts to produce such vaccine against Babesia rossi infection using SPA from B. rossi culture supernatants were not or only partially successful. Here we show that when dogs were vaccinated with a vaccine comprising SPA from B. rossi combined with SPA from Babesia canis protective immunity against experimental challenge infection was induced. Immunity was reflected in reduced clinical signs that resolved spontaneously, and reduction of parasitaemia and SPA in the blood. Not a single infected erythrocyte could be found in blood smears of dogs that had been repeatedly boosted (three vaccinations in total). In contrast, three out of four control dogs required chemotherapeutic treatment to prevent death. The fourth control dog showed a transient parasitaemia that resolved spontaneously. Vaccination did not prevent the development of a transient anaemia. It is concluded that a vaccine containing a mixture of SPA obtained from in vitro culture supernatants of B. rossi and B. canis induces protection in dogs against heterologous challenge infection with B. canis (as shown before) or B. rossi.

The complete set of Toxoplasma gondii ribosomal protein genes contains two conserved promoter elements.

Recently we showed that de novo ribosome biosynthesis is transcriptionally regulated in Coccidia, depending on their life-cycle stage. Since the expression of ribosomal protein genes is likely coordinated, the transcriptional control of all Toxoplasma gondii ribosomal protein (RP) genes was analysed. Therefore, the complete set of all cytoplasmic RPs was defined, containing 79 different RPs in T. gondii. RP genes were randomly distributed over the genome, each with a unique upstream region with the exception of 8 RP genes which were paired in a head-to-head orientation. To study if the RP genes share conserved promoter elements, a database was made containing upstream sequences of all T. gondii RP genes. Promoter activity was confirmed for the upstream sequences of 8 RP genes, some of which are comparable in strength to the alpha-tubulin promoter. In the complete set of RP upstream sequences 2 novel and highly conserved elements were identified, named Toxoplasma Ribosomal Protein (TRP)-1 (consensus: TCGGCTTATATTCGG) and TRP-2 ([T/C]GCATGC[G/A]). TRP-1 and/or TRP-2 were present in 95% of all RP upstream sequences and moreover, were specifically localized in a small region near the presumptive transcriptional start site (10-330 bp upstream). Although TRP elements were mostly absent in known T. gondii promoters, they are present elsewhere in the T. gondii genome suggesting that they operate not only in RP genes but in a larger set of genes. The identification of TRP elements creates a basis to further study the underlying mechanism by which RP transcription is controlled in T. gondii.

Onset and duration of immunity against Babesia canis infection in dogs vaccinated with antigens from culture supernatants.

It has previously been shown that dogs can be vaccinated against heterologous Babesia canis infection using a vaccine containing soluble parasite antigens (SPA) from in vitro cultures of B. canis and B. rossi that are adjuvanted with saponin. In the present study the onset and duration of immunity of vaccinated dogs were studied. Results showed that 3-26 weeks after initial vaccination, dogs effectively limit the level of SPA in plasma upon challenge infection, which was reflected in limited duration and extent of clinical manifestations. There was no statistically significant effect of vaccination on the parasite load in the circulation, which was determined from blood smears. It was further shown that the level of immunity of primary vaccinated dogs (priming and booster vaccination with a 6-week interval) and that of repeatedly vaccinated dogs (a single additional vaccination 6 months after primary vaccination) is comparable. From this study it is concluded that vaccination with this preparation induces protective immunity against clinical babesiosis from 3 weeks after booster vaccination onwards, and remains effective for a period of at least another 6 months. A single booster vaccination is sufficient to maintain immunity for at least another 6 months.

Vaccination with a preparation based on recombinant cysteine peptidases and canine IL-12 does not protect dogs from infection with Leishmania infantum.

Cysteine peptidases (CPs) have been implicated in various processes central to the pathogenicity of Leishmania parasites, and are thought to be key factors in the host-parasite interaction. In order to fully evaluate the potential of the CPs as vaccine candidates, studies in natural host species are required. In the study we report here, recombinant L. infantum CPs CPA and CPB were used to vaccinate dogs. In order to induce an appropriate response against the antigens, recombinant canine IL-12 was added as an adjuvant either by itself or in combination with Quil A. After vaccination, dogs were given an intravenous challenge with promastigotes of L. infantum JPC strain. In both vaccinated groups (CPs with IL-12 or CPs with IL-12 and Quil A) CP-specific antibodies were detected after vaccination, indicating that there was a reaction to the vaccine. However, all dogs were found parasite-positive and all developed some degree of clinical leishmaniosis. The observed lack of efficacy of the candidate vaccines could be due, completely or in part, to a number of factors associated with the vaccine antigen, the adjuvant or host-parasite interactions. When compared to results from other studies, it seems less likely that the molecular conformation of the rCPs or rIL-12 caused this lack of efficacy. More plausible explanations are the dose and timing of the IL-12 application and the potentially different effects IL-12 induces as an adjuvant in either the murine or the canine leishmaniosis model.

Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora.

The protozoan parasites Eimeria spp. Toxoplasma gondii and Neospora caninum are significant causes of disease in livestock worldwide and T. gondii is also an important human pathogen. Drugs have been used with varying success to help control aspects of these diseases and commercial vaccines are available for all three groups of parasites. However, there are issues with increasing development of resistance to many of the anti-coccidial drugs used to help control avian eimeriosis and public concerns about the use of drugs in food animals. In addition there are no drugs available that can act against the tissue cyst stage of either T. gondii or N. caninum and thus cure animals or people of infection. All three groups of parasites multiply within the cells of their host species and therefore cell mediated immune mechanisms are thought to be an important component of host protective immunity. Successful vaccination strategies for both Eimeria and Toxoplasma have relied on using a live vaccination approach using attenuated parasites which allows correct processing and presentation of antigen to the host immune system to stimulate appropriate cell mediated immune responses. However, live vaccines can have problems with safety, short shelf-life and large-scale production; therefore there is continued interest in devising new vaccines using defined recombinant antigens. The major challenges in devising novel vaccines are to select relevant antigens and then present them to the immune system in an appropriate manner to enable the induction of protective immune responses. With all three groups of parasites, vaccine preparations comprising antigens from the different life cycle stages may also be advantageous. In the case of Eimeria parasites there are also problems with strain-specific immunity therefore a cocktail of antigens from different parasite strains may be required. Improving our knowledge of the different parasite transmission routes, host-parasite relationships, disease pathogenesis and determining the various roles of the host immune response being at times host-protective, parasite protective and in causing immunopathology will help to tailor a vaccination strategy against a particular disease target. This paper discusses current vaccination strategies to help combat infections with Eimeria, Toxoplasma and Neospora and recent research looking towards developing new vaccine targets and approaches.

An Eimeria vaccine candidate appears to be lactate dehydrogenase; characterization and comparative analysis.

An Eimeria acervulina protein fraction was identified which conferred partial protection against an E. acervulina challenge infection. From this fraction a 37 kDa protein was purified and its corresponding cDNA was cloned and shown to encode a lactate dehydrogenase (LDH). Full length cDNAs encoding LDH from two related species, E. tenella and E. maxima, were also cloned. The homology between the primary amino acid sequences of these three Eimeria LDH enzymes was rather low (66-80%), demonstrating an evolutionary divergence. The Plasmodium LDH crystal structure was used to generate a 3D-model structure of E. tenella LDH, which demonstrated that the many variations in the primary amino acid sequences (P. falciparum LDH and E. tenella LDH show only 47% identity) had not resulted in altered 3D-structures. Only a single LDH gene was identified in Eimeria, which was active as a homotetramer. The protein was present at similar levels throughout different parasitic stages (oocysts, sporozoites, schizonts and merozoites), but its corresponding RNA was only observed in the schizont stage, suggesting that its synthesis is restricted to the intracellular stage.

Control of coccidiosis in chickens by vaccination.

Control of coccidiosis in chickens has relied upon managerial measurements and the prophylactic use of coccidiostatic drugs. With the emergence of Eimeria strains that are resistant to these drugs the use and number of commercially available vaccines has increased. In this review various aspects that contribute to the development of coccidiosis are discussed, and an overview of the currently marketed coccidiosis vaccines is presented. Three groups of vaccines can be distinguished based on the characteristics of the Eimeria species included in the products: vaccines based on live virulent strains, vaccines based on live attenuated strains, and vaccines based on live strains that are relatively tolerant to the use of ionophores. These latter vaccines combine the early protective effect of ionophore treatment with the late protective effect of vaccination. The impact of future developments such as recombinant-DNA vaccines and changes in consumer's attitude towards broiler production are discussed.

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants.

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.

Vaccination against Eimeria tenella infection using a fraction of E. tenella sporozoites selected by the capacity to activate T cells.

At 8 days after a primary Eimeria tenella infection, a subset of T cells, of which the protective role is as yet unclear, circulates in the peripheral blood. In order to investigate this, the in vitro cellular responsiveness of these peripheral blood lymphocytes has been used as selection criterion to identify potentially protective E. tenella sporozoite antigens. The hydrophilic protein phase of purified E. tenella sporozoite homogenates obtained by Triton X-114 extraction was fractionated using preparative gel electrophoresis. Nine fractions, separated according to different molecular weight, were tested for their ability to stimulate T-cell responses. Both the proliferation of peripheral blood lymphocytes and the macrophage activating activity released in the culture supernatants were measured. On the basis of this responsiveness, four fractions were selected and used to vaccinate chickens. All vaccine preparations induced strong T-cell responses. One fraction immunised chickens against subsequent challenge infection, in that the caecal lesion scores were significantly lower as compared with that of the unvaccinated controls. This fraction contained hydrophilic polypeptides with a molecular mass that ranged from 26 to 30 kDa.

Vaccination of broilers with HVT expressing an Eimeria acervulina antigen improves performance after challenge with Eimeria.

The sequence encoding part of the 100 kDa refractile body protein (Ea1A) from Eimeria acervulina was cloned into the US10 locus of herpesvirus of turkeys (HVT) downstream of LTR promoter. Expression of the fusion protein was shown in vitro. Recombinant HVT showed a delayed and slightly reduced level of viremia compared to the parent strain in SPF chickens as well as in broilers. Effect on the performance of broilers vaccinated with recombinant or parent HVT was measured by challenge at day 24 with a high dose of E. acervulina and E. maxima oocysts. A significant improvement in weight of animals vaccinated with the recombinant HVT was detected at the end of the challenge period.

Progress in recombinant vaccine development against coccidiosis. A review and prospects into the next millennium.

The increasing problems encountered by the poultry industry, despite the extensive use of drugs, have emphasised the need for an immunological solution for the economic damage caused by the Eimeria parasite. Although immunity develops relatively fast following a natural infection, to induce protection by using parasite extracts or single antigens appears more difficult. Nevertheless, the development of a vaccine based on defined antigens seems the best solution in the long run. At the VIth International Coccidiosis Conference in 1993 the first promising results were reported from small-scale experiments using recombinant antigens. This review summarises the advances in this field of research from 1993 onwards. Although since then not many reports have been published about the effects of using recombinant. antigens as a vaccine against coccidiosis, a number of interesting new proteins which could be considered good targets for such a vaccine have been described and are referred to herein. Proteins involved in the process of invasion of the host cell by the extracellular parasite are regarded as key components in the developmental cycle of the parasite. These components possibly bind to receptors on the host cell. Interference with this process could be a target of the protective immune response. Progress has also been made in characterising the immune mechanisms activated by infection with the parasite. From experimental mouse models and from studies in chickens, a better insight has been obtained towards the involvement of CD4- and CD8-type T cells in, respectively, the inductor and the effector branch of the immune response, although not all questions have been answered. Several antigens have been selected using T-cell stimulation and cytokine assays and these are reviewed. In a third section, mostly unpublished results of our own experiments dealing with the use of live vectors to present defined antigens such as Ea1A and EaSC2, a parasite refractile body transhydrogenase and a lactate dehydrogenase, respectively, are summarised. Partial protection could be induced using Salmonella typhimurium as a carrier for these antigens, in that the oocyst output was reduced by up to 50% after challenge and weight gain could be improved by 5-10% over non-vaccinated challenged chickens, when tested in a floor-pen trial. Similar results were obtained when these antigens were presented by viral vectors such as Fowlpox virus or Herpes virus of turkey. These data seem to offer good prospects for the accomplishment of a safe and efficaceous vaccine based on recombinant DNA technology. These expectations are corroborated by recent breakthrough in transfection of related parasites such as Plasmodium and Toxoplasma gondii, and by the increasing amount of genomic information becoming available every day, the impact of which cannot even be estimated yet. These new technologies will allow us to solve the complex problems that we once created ourselves.

Induction of a local and systemic immune response using cholera toxin as vehicle to deliver antigen in the lamina propria of the chicken intestine.

In this study, the humoral mucosal immune response to a recombinant Eimeria antigen (Ea1A) was enhanced using cholera toxin (CT). Chickens were primed intra-intestinally with Ea1A either conjugated or not to CT. The local and systemic antibody responses to both Ea1A and CT were determined to find out whether the chickens could respond to CT and whether both antigens had reached the lamina propria. In addition the effects of CT on lamina propria leukocytes were examined. The results showed that chickens had receptors on the caecal epithelium that could bind CT. At day 7 after administration, the number of CD4+ and CD8+ T lymphocytes in the lamina propria of the caecum had increased, indicating that CT had a specific immunological effect. At this timepoint, anti-CT antibody containing cells were detected locally in the lamina propria of the caecum. In serum all antigen preparations containing CT induced IgM and IgG antibody titres specific for CT within 10 days after priming. In addition, the recombinant Ea1A antigen also induced serum responses when administered together with CT or conjugated to CT, thus both CT and the antigen had reached the lamina propria. Nevertheless, the Ea1A specific response was much higher in the primary response and after booster immunization when the antigen was conjugated to CT than when only mixed with CT. Therefore, we conclude that CT is a suitable adjuvant for intra-intestinal application in chickens, especially when the antigen is conjugated to it.

Peripheral blood lymphocytes from Eimeria tenella infected chickens produce gamma-interferon after stimulation in vitro.

Protective immunity to infection by Eimeria parasites has been demonstrated to be dependent on T-cell mediated immune responses and may be associated with the release of cytokines. We have previously shown that the proportion of CD8-expressing T-cells in the peripheral blood of chicken increases transiently at 8 days after a primary infection with Eimeria tenella oocysts. The increase in the CD8+ population coincided with an increased proliferative lymphocyte response upon stimulation with E. tenella sporozoite antigen in vitro. In this study, we further investigated the functional activity of these peripheral blood leucocytes (PBL) by determining both the potential to proliferative and to produce IFN upon stimulation with E. tenella sporozoite antigens and mitogens. Enhanced proliferative responses to parasite antigen were accompanied by reduced responses to T-cell mitogens around 1 week of infection. The IFN activity in the supernatants of the stimulated PBL was measured by the ability to inhibit Semliki Forest Virus (SFV) replication in chicken embryo fibroblasts (CEF) and to activate macrophages, as measured by nitric oxide production. At eight days after infection the highest levels of virus inhibition and NO-production were detected upon stimulation with both E. tenella sporozoite antigen and mitogen. A strong correlation between the individual data of the two methods was found at this timepoint indicating that the produced cytokine was indeed IFN-gamma. These results suggest that around eight days after a primary E. tenella infection a parasite specific T-cell subset with the capacity of produce IFN(-gamma) is circulating which would be involved in the induction of protective immunity against Eimeria tenella.

Characterization of phenotype related responsiveness of peripheral blood lymphocytes from Eimeria tenella infected chickens.

We have previously shown that the proportion of CD8-expressing T cells (CD8bright+ and CD4+ CD8dim+ cells) in the peripheral blood of chickens increases around 8 days after a primary infection with Eimeria tenella oocysts. The increase in the CD8+ eight cells coincides with enhanced responses after in vitro stimulation with parasite antigen. In the study described here, the responsiveness of these day 8 PBL was further characterized by determining their capacity to proliferate and to produce cytokine (IFN-gamma) upon stimulation with E. tenella sporozoite antigen, or non-specific stimuli like T cell growth factor (TCGF) and anti-CD3 monoclonal antibody (MoAb). Comparing the responsiveness of infected responder (day 8) and control chickens, non-specific triggering induced cytokine production in cells from infected animals and proliferation in cells from control animals. When triggered with E. tenella sporozoite antigen, lymphocytes from infected chickens responded with proliferation and cytokine production, in contrast to lymphocytes from control animals that did not respond. The phenotype of the lymphocytes involved in the parasite-specific proliferation and cytokine production, was characterized in a blocking assay using MoAb directed against the CD4 or CD8 molecule. The results suggest that CD8bright+ as well as CD4+ (CD4+ CD8dim+ and possible CD4+, single positive) lymphocytes are responsible for the IFN-gamma production measured after stimulation with parasite antigen, whereas the specific proliferative response appears to be caused by CD4+ (CD4+ CD8dim+ and possibly CD4+ single positive) lymphocytes. We speculate that the CD8bright+ cells, present in the circulation around 8 days after a primary E. tenella infection, act as effector cells in protective immune responses, whereas CD4+ cells play an important helper function in these responses.

Eimeria tenella infections in chickens: aspects of host-parasite: interaction.

Intestinal coccidiosis, caused by various species of Eimeria, has become an economically important disease of poultry and livestock throughout the world. Infection of chickens starts after ingestion of oocysts when sporozoites penetrate the epithelium of the villi. After passage through the lamina propria, they enter crypt epithelial cells where they undergo several rounds of asexual and sexual proliferation, thus forming merozoites and later, gametocytes. When macrogametes are fertilized by microgametes, oocysts are formed that are shed in the faeces. Nowadays, coccidiosis is prevented by anticoccidial drugs that are added to food, but the prolonged use of these drugs leads inevitably to the emergence of resistant Eimeria strains. During infection, there are three stages when the chicken immune system can inhibit parasitic development. The first is when the sporozoite searches for a site of penetration and binds to the epithelium. The second is when the sporozoite is in the villus epithelium amongst intra-epithelial leucocytes. The third is during its passage through the lamina propria to the crypt epithelium. To investigate this, the decisive factors in the induction and effector phase of immunity against coccidiosis have been investigated in situ. Our studies have revealed that three phenomena are responsible for immunity against Eimeria infections. First, the actual passage and presence of parasites in the lamina propria to induce immunity. Second, the sporozoite seems to be the most important parasite stage for immunity, and third, cytotoxic T cells are necessary to inhibit parasites.

Differential binding of two monoclonal antibodies directed against the chicken CD8 alpha molecule.

Two murine monoclonal antibodies (mAb), CT8 and AV14, have been shown to recognise the avian homologue of the mammalian CD8 alpha molecule. In previous flow cytometry studies we could discriminate two subpopulations of CD8+ T cells, expressing either a high level (CD8Bright+) or a low level (CD8Dim+) of CD8 molecules. The staining patterns of mAb AV14 and mAb CT8 were not always identical for individual chickens. In this study the discrepancy in the reactivity of these mAb was examined, using outbred White Leghorn chickens as well as (B14B14)-MHC inbred Wellcome chickens. The results show that mAb AV14 and mAb CT8 recognise different epitopes on the chicken CD8 alpha molecule. The CD8Bright+ cells appeared to express the CD8 alpha beta heterodimer and the CD8Dim+ cells the CD8 alpha alpha homodimer. Conformational differences between the alpha beta heterodimer and the alpha alpha homodimer could account for the differences in binding characteristics found for the two mAb. The existence of a polymorphism of the CD8 alpha molecule in outbred White Leghorn chickens was suggested by the failure of peripheral blood leucocytes from some chickens to react with mAb AV14. This heterogeneity was not observed in the Wellcome line.

In situ characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens.

We characterized the leucocyte subpopulations after infection with Eimeria tenella in both naive and immune chickens. Immunocytochemical staining was used to characterize the cells in situ, so that the interaction between host and parasite could be studied. More leucocytes were detected in the lamina propria of immune chickens, and leucocytes infiltrated the ceca more rapidly than in naive chickens, but the infiltration was less pronounced than in naive chickens. In naive chickens, most infiltrated leucocytes were macrophages and T cells. Two days after inoculation the number of CD4+ cells had increased greatly. In immune chickens, mainly T cells (CD4+ and CD8+) infiltrated the lamina propria, and in contrast to naive chickens, the number of CD8+ cells exceeded the number of CD4+ cells. Furthermore, we characterized which cells contained a parasite and which cells were detected next to the parasites, because these cells are probably involved in the arrested development of the parasites. In naive chickens, sporozoites were significantly more often located within or next to macrophages than in immune chickens. In immune chickens, sporozoites were significantly more often located within or next to CD3+, CD8+, and TCR2+ cells. In conclusion, the marked increase of CD4+ cells after primary infection suggests that these cells are involved in the induction of the immune response, whereas the increase of CD8+ cells after challenge infection suggests that these cells act as effector cells.

Immunity to eimeria tenella in chickens: phenotypical and functional changes in peripheral blood T-cell subsets.

The changes in peripheral blood leukocyte (PBL) T-cell subsets following Eimeria tenella infection in outbred white leghorn chickens were studied, using a panel of murine monoclonal antibodies specific for the chicken homologues of the mammalian CD3, CD8, and CD4 markers on day-to-day samples of PBLs. Both flow cytometric analysis (FCA) and immunofluorescence microscopy with fixed cells on slides were used as read-out systems. The changes in the composition of the T-cell subsets measured with both techniques were similar. At 8 days post primary infection, a sharp transitory increase in the proportion of CD8-expressing cells was found. With FCA, CD8-expressing cells could be discriminated in CD8(Dim+) and CD8(Bright+) populations, which have not been described before. The proportion of CD4-expressing cells was decreased at days 9-10 after primary infection, which coincided with a less marked decrease in CD3-expressing cells. Such effects were not seen after secondary infection. When PBLs collected at day 8 post primary infection were stimulated in vitro with E. tenella sporozoite antigen, the response was higher than that in uninfected control chickens. The effects we observed coincide with the onset of recovery from primary infection. We speculate that the increase in CD8-expressing PBLs is the result of stimulation and expansion of a specific subset involved in the induction of protective immunity against Eimeria tenella.

Eimeria tenella: sporozoites rarely enter leukocytes in the cecal epithelium of the chicken (Gallus domesticus).

In this study, the intraepithelial leukocytes supposedly involved in the transportation of Eimeria tenella sporozoites through the cecal lamina propria were phenotypically characterized. The ceca of naive and immune chickens were examined at various times after inoculation by light microscopy and immunocytochemical techniques. The distribution of sporozoites within the villus differed markedly between both groups. From 16 hr postinoculation, significantly fewer sporozoites had reached the crypts in immune chickens, and schizont formation was inhibited. In the villus epithelium of both naive and immune chickens, few sporozoites were found within a leukocyte. Using anti-sporozoite and anti-CD45 monoclonals, we showed that even when intraepithelial leukocytes are abundantly present, only a few sporozoites were inside them. In the lamina propria of immune chickens significantly more sporozoites were found within leukocytes than in the lamina propria of naive chickens. The phenotype of the few leukocytes that harbored sporozoites was similar in naive and immune chickens. A few sporozoites were detected in B cells, 10% in macrophages, and 50% in T cells, especially CD8+ cells. These results show that E. tenella sporozoites rarely enter intraepithelial leukocytes and therefore their putative role in transporting sporozoites through the lamina propria is doubtful.