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Infection and Immunity, October 2007, p. 4909-4916, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00710-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Influence of Host Immunity on Parasite Diversity in Theileria parva

Frank Katzer,¹ Daniel Ngugi,¹ Christian Schnier,² Alan R. Walker,¹ and Declan J. McKeever^1,2*

Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, United Kingdom,¹ Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, United Kingdom²

Received 25 May 2007/ Returned for modification 26 June 2007/ Accepted 9 July 2007

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We examined the influence of host immunity on the genotypic diversity of the intracellular transforming cattle parasite Theileria parva. By tracking the emergence of discrete parasite genotypes in an animal challenged with a bulk stabilate following immunization with its major component clone, we observed a profound modulation of genotypic frequencies in the breakthrough schizont population. In particular, no incidences of the immunizing clone were observed and a progressive decline was apparent in the relatedness of breakthrough genotypes to it. These observations were reflected in the genotypic profile of transmissible parasite stages that emerged in the erythrocyte fraction of the animal and in parasite progeny generated by tick pickup. In a separate experiment, genotypic profiles of breakthrough parasite populations were observed to vary between unrelated immune animals selected on the basis of the major histocompatibility complex (MHC) class I phenotype, a known determinant of the specificity of the immune response. Furthermore, immunization and challenge of calves with molecularly distinct but cross-protective parasite populations revealed that infection results in transmissible erythrocyte forms in spite of a protective immune response. These observations suggest that immunity does not prevent transmission of challenge parasites and that its impact on the parasite at a population level is influenced by herd MHC diversity.

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The genetic structure of a pathogen population is determined on the one hand by its potential for generating diversity and on the other by selective influences that limit that diversity. For many pathogens, a major driver for selection, and hence a significant determinant of population structure, is the host immune response. For pathogens that are transmitted by vector species, the population structure is expected to reflect pressures from both host and vector defenses. For example, while there is ample evidence for immune selection against Plasmodium falciparum in humans, substantial attrition of malaria parasites also occurs during differentiation in the mosquito vector (28). Considerable attrition (in the region of 4 to 5 orders of magnitude) is also observed during passage of Theileria parva, a related parasite of cattle, through its tick vector, Rhipicephalus appendiculatus (13). However, little is known about the impact of immune selection in the bovine host on the population structure of this parasite, which causes an often fatal lymphoproliferative disease known as East Coast fever in eastern, central, and southern Africa (12).

The life cycle of T. parva is largely asexual, with only a brief sexual phase in the tick vector (17). Sporozoites inoculated by infected ticks rapidly invade host lymphocytes, where they differentiate into multinucleate intracellular schizont forms capable of activating the infected cell and preventing apoptosis (3, 11). This results in uncontrolled proliferation, with the parasite dividing in synchrony with the cell and redistributing to each daughter cell during cytokinesis. The pathology associated with East Coast fever arises from invasion of lymphoid and nonlymphoid tissues by parasitized lymphoblasts. In a proportion of infected cells, the parasite undergoes further differentiation into uninucleate merozoite forms, which, upon release from the dying cell invade erythrocytes and develop into piroplasms, the infective stage for ticks. When ingested by a subsequently feeding tick, piroplasms give rise to gametes, which undergo syngamy in the gut lumen to form zygotes. These invade gut epithelial cells and differentiate into motile kinetes, which enter the hemocoel and migrate to the salivary gland. There, they invade specialized acinar cells and undergo further schizogonous division to form sporozoites.

Immune mechanisms deployed by cattle against T. parva have been well characterized (15, 20). With the exception of animals that have been repeatedly challenged, little or no response is observed against sporozoite antigens (22). Rather, the principal target of the response is the schizont-infected lymphoblast, as evidenced by the appearance of parasite-specific CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) in the peripheral blood of immune cattle 5 to 7 days after infection, when schizonts are first detected (21). These CTL are highly effective at killing infected cells in vitro, and their appearance is associated with reduction of schizont parasitosis to undetectable levels. Their fundamental role in immunity to T. parva has been demonstrated by the transfer of immunity between immune and naïve calves in the CD8⁺ fraction of lymph derived from a node responding to challenge (16). Several lines of evidence suggest that the T. parva-specific CTL response is rather narrow in its focus. It is almost invariably restricted by only one of the parental MHC haplotypes, and where multiple class I products are expressed from a given haplotype, only one restricts the response (19, 30). Furthermore, certain parasite determinants appear to be immunodominant in the context of individual class I products. This observation has emerged from studies of the Muguga and Marikebuni stocks of the parasite. These have a nonreciprocal cross-reactivity profile such that immunization with Marikebuni invariably protects against Muguga, while only a small proportion of Muguga-immunized animals resist challenge with Marikebuni. Hence, in the majority of Muguga-immunized cattle, strain-specific determinants are dominant, whereas cross-reactive determinants are preferentially targeted by animals exposed to Marikebuni (30). When they are observed in Muguga-immunized animals, cross-reactive responses appear to recognize a number of distinct determinants within the Marikebuni stock, with the MHC phenotype of the animal determining which is targeted (8).

A major uncertainty with respect to the CTL response against T. parva is the extent to which it imposes selective pressure on the parasite at the population level. This would require a significant impact on transmission through prevention of schizont differentiation to merozoites, which are the precursors of tick-infective piroplasms. It is not clear that the kinetics of the CTL response are sufficiently rapid to eliminate a given schizont population prior to the emergence of merozoites. Schizont-infected cells are commonly seen in immune cattle after challenge, and there is evidence from studies of the related parasite Theileria annulata that infected cells vary in the efficiency with which they undergo merogony (26, 27). Hence, it is possible that a proportion of cells infected with a given parasite genotype undergo differentiation before being eliminated by the CTL response. In addition, since piroplasms of T. parva undergo only limited replication (2), it is believed that the carrier state in recovered immune cattle is maintained by small numbers of schizont-infected cells that persist in spite of the response.

Using a genome-wide panel of polymorphic markers, we have recently provided evidence for extensive genotypic diversity in T. parva populations derived from ticks fed on cattle infected with the Marikebuni stock of the parasite (13). Substantial reassortment of alleles at marker loci was evident on additional calf/tick passage, which is consistent with frequent recombination and the capacity to respond to selective pressures imposed by host or vector. Here, we describe the application of this genotyping approach to evaluate the effects of the CTL response on further differentiation of the parasite in the bovine host and on the population structure of the parasite following transmission through the tick vector.

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Parasite populations. Sporozoite stocks were derived from the Muguga and Marikebuni isolates of T. parva. The former was isolated in Kenya in the 1930s and is essentially homogeneous following prolonged laboratory passage. The Muguga stock used in the present study was Centre for Tropical Veterinary Medicine (CTVM) stabilate 71 (St71). The Marikebuni isolate was obtained in the 1980s (18) and, having undergone relatively few passages, is considerably more heterogeneous than Muguga. The CTVM St72 stabilate was used and represents the fifth passage of the isolate from the field. St72 is dominated by a genotype designated 72-01, which accounts for approximately 75% of the population (13). A third stabilate, St103, was used that was derived from R. appendiculatus ticks fed on an animal infected by inoculation with 5 x 10⁶ autologous parasitized cells carrying a 72-01 clone.

Animal infections. Calves were infected by subcutaneous inoculation with sporozoite stabilate above the right prescapular lymph node. For immunization, the animals were simultaneously administered a long-acting formulation of oxytetracycline (Terramycin/LA; Pfizer) at a dose of 20 mg/kg of body weight. For some experiments, calves were immunized by subcutaneous inoculation with 5 x 10⁶ in vitro-infected autologous cells. Infections are generally mild under these circumstances, and the animals were treated with oxytetracycline (Engemycin; 20 mg/kg; Intervet) only in the event of persistent schizont parasitosis and fever. The progress of infection was monitored by daily evaluation of rectal temperature and detection of T. parva schizonts by immunofluorescence or Giemsa staining of lymph node needle aspirates taken on alternate days after the first manifestation of fever. The presence of piroplasm forms was evaluated through analysis of Giemsa-stained blood smears. With the exception of immunization experiments, calves were euthanized on days 18 to 21 of infection or, where ticks had been applied, after detachment of all engorged nymphs (see below). All animal experiments were conducted in compliance with the United Kingdom Animals (Scientific Procedures) Act 1986.

Where appropriate, transmission of infection to ticks after challenge was accomplished by application of cloth bags containing approximately 1,000 R. appendiculatus nymphs to the ears of the calf at the indicated time points postinfection. Engorged nymphs were collected each day and allowed to molt by incubation for 4 weeks at 28°C and 85% relative humidity. After prefeeding on rabbits for 4 days to stimulate sporogony, the molted adults were surface sterilized by sequential rinses in 5% chlorhexidine, 70% ethanol, and antibiotics. A sample was removed prior to sterilization for assessment of the prevalence and abundance of infection in salivary gland smears stained with methyl green and pyronin (32). Stabilates were prepared by trituration of sterilized ticks essentially as described by Brown (1).

Preparation of cellular fractions and DNA for marker analysis. Approximately 800 µl jugular venous blood collected in Alsever's solution was overlaid on an equal volume of Ficoll-Paque and centrifuged at 900 x g for 30 min. The plasma, mononuclear cell, and Ficoll layers were discarded, along with the top of the red blood cell (RBC) layer, leaving an RBC pellet of approximately 100 µl. DNA was extracted from this pellet using the QIAmp DNA blood minikit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions.

Lymph node aspirates were collected in 500 µl Alsever's solution and centrifuged at 5,000 x g for 2 min before the supernatant was discarded. DNA was then extracted from the cell pellets using the Wizard SV genomic-DNA purification system (Promega Corporation, Madison, WI) following the manufacturer's instructions.

Genotypic analysis. Multilocus genotypes were compiled for individual parasite clones derived by in vitro infection and cloning as described previously (13). Genotypes were determined by PCR amplification of a panel of 64 polymorphic loci comprising minisatellite and microsatellite markers and genes with known size polymorphisms. Precise information on the marker panel is available in Katzer et al. (13), along with details of the PCR conditions.

CTL assays. Induction of CTL activity following immunization was confirmed by evaluating the in vitro cytotoxic activity of peripheral blood mononuclear cells (PBMC) on ⁵¹Cr-labeled autologous infected lymphoblasts. PBMC were prepared from jugular venous blood collected in Alsever's solution by flotation on Ficoll-Paque as described by Goddeeris et al. (5) and restimulated by coculture with irradiated autologous infected cells. Killing activity was then determined in 96-well plates using a 4-h chromium release assay. Supernatants from individual wells were harvested and assessed for activity in a gamma counter. The precise conditions for restimulation and cytotoxicity assays were those described by Goddeeris and Morrison (6).

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Influence of the CTL response on targeted genotypes. To determine the extent to which parasite populations targeted by the CTL response are compromised during infection, a naïve 9-month-old male Friesian calf (no. 211) was immunized against the dominant 72-01 genotype of St72 by inoculation with 5 x 10⁶ autologous cells (representative of the different lymphocyte lineages) infected with the clone. The animal was treated on days 9 and 10 after infection with 20 mg/kg oxytetracycline (Engemycin; Intervet), and unfed nymphal R. appendiculatus ticks were applied on days 9 and 11 with the purpose of generating a sporozoite stabilate of the genotyope as described above. The resulting stabilate was designated St103. The animal recovered uneventfully and was confirmed to have developed CTL specific for the immunizing parasite (Fig. 1).

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FIG. 1. Recognition of the T. parva genotype 72-01 by calf 211 lymphocytes after three in vitro restimulations with autologous 72-01-infected lymphoblasts. The values depict effector-induced 51Cr release from labeled target cells as a percentage of total release (as achieved by freeze-thaw lysis) over a range of effector/target ratios. , autologous targets; , MHC class I -matched targets; , MHC-mismatched targets.

Eight weeks after immunization, the calf was challenged with 1.25 tick equivalents of the parent stabilate, St72. The progress of infection was monitored as described above, and unfed nymhal ticks were applied (see below) in order to transmit the breakthrough parasite population for genotypic analysis. The animal developed a fever (as defined by a temperature of 39.5°C) on day 7 after infection that persisted until day 13. Macroschizonts were detectable in the draining lymph node from day 8 until day 15, by which time the calf had recovered. Piroplasms were not apparent in blood smears until day 17 and were still present on day 18, when the remaining engorged nymphs were collected and the animal was euthanized.

Sterile needle aspirates were collected from the draining lymph node at periodic time points after infection. Aspirated cells collected on days 9, 14, and 16 were purified by flotation on Ficoll-Paque as described previously (5), and parasitized cells were cloned by limiting dilution in the presence of gamma-irradiated autologous PBMC as described by Katzer et al. (13) and subjected to genotypic analysis with the marker set. In total, 162 clones were examined, none of which bore the immunizing 72-01 genotype or any other previously observed in St72. This contrasts with earlier observations in a naïve animal infected with St72, where approximately 75% of clones derived from lymph node aspirates bore the 72-01 genotype (13). Eleven genotypes were observed among 62 clones derived from the day 9 aspirate, and one, designated 211-A3, differed from the 72-01 genotype at only one locus (ms010) (24). The day 14 aspirate yielded 95 clones, among which 10 distinct genotypes were observed, while 3 genotypes were detected among 5 clones isolated from the day 16 aspirate. A Treeview phylogram (25) derived from a pairwise distance matrix generated, using the LIAN 3.1 software (10), from the 211 lymph node aspirate data and that previously obtained for St72 (13) is presented in Fig. 2 and suggests that relatedness of breakthrough genotypes to the immunizing clone declined with the progress of infection. This is illustrated more clearly by a boxplot (Fig. 2, inset) generated by the SAS statistical package from genotypic similarities of breakthrough parasites to the immunizing clone. Comparison of values for genotypes observed on days 9 and 14 using a Wilcoxon two-sample test yielded a one-sided exact P value of 0.0007, indicating a significantly lower (P < 0.05) level of relatedness on day 14.

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FIG. 2. Treeview phylogram derived from a pairwise distance matrix generated from St72 and calf 211 lymph node breakthrough genotypes using the LIAN 3.1 software. Breakthrough genotypes isolated from calf 211 on days 9, 14, and 16 of the challenge infection are prefixed A, B, and C, respectively. The immunizing 72-01 genotype is indicated by the arrow. (Inset) Boxplot of genotype similarity values (percentages of alleles shared) for calf 211 breakthrough clones with respect to the immunizing 72-01 clone on the indicated days after challenge. Day 14 values are significantly lower (P < 0.05) than those calculated for day 9, as revealed by a Wilcoxon two-sample test (one-sided exact P = 0.0007).

Kinetics of the emergence of breakthrough populations. To examine the kinetics of infection with discrete components of St72 following challenge, whole DNA prepared from individual lymph node aspirates collected during the course of infection was analyzed with selected markers that distinguish the immunizing 72-01 genotype. The results of one such analysis, using the TP04_0051 marker, are presented in Fig. 3. This marker, also known as the polymorphic immunodominant molecule (31), is characterized by a central region with various numbers of repeat sequences that result in distinct amplicon sizes. Three alleles were found among the genotypes obtained from St72 (13), with approximate sizes of 850, 791, and 550 bp. The 791-bp allele is the one carried by the 72-01 genotype (data not shown). Three alleles were apparent in the day 7 aspirate: the 791-bp product characteristic of the 72-01 genotype and an additional 750-bp allele were prominent, while the 550-bp band was only faintly discernible. The 791-bp allele was seen to decline markedly over the remainder of the sampling period and was completely absent beyond the day 15 sample. In contrast, the intensity of the 550-bp allele increased dramatically after day 9, while the novel 750-bp product remained prominently expressed throughout the time course. The day 18 aspirate contained only the 750-bp and 550-bp alleles, with the former being the dominant species. Hence, the infection at the lymph node level was characterized by elimination of the dominant 791-bp allele carried by the immunizing genotype and a corresponding expansion of the minor 550-bp allele, along with an additional, previously unencountered allele of 750 bp.

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FIG. 3. Emergence of parasite genotypes in lymph node and erythrocyte compartments of calf 211 on the indicated days after challenge with St72, as revealed by PCR amplification of the TP04_0051 polymorphic marker. The arrowhead indicates the allele carried by the immunizing 72-01 clone.

The lymph node analysis is effectively informative only at the level of the schizont-infected lymphoblast. To evaluate the kinetics of infection with the piroplasm stage of the parasite, DNA was prepared from erythrocytes isolated from peripheral blood during the course of infection and subjected to marker analysis. Parasite products were first observed in this fraction on day 12 after infection, when the 791-bp allele associated with the 72-01 genotype and the 550-bp alleles were apparent as weak bands. Both alleles were prominent in the day 14 sample, along with a faint band at 750 bp. Although the 550-bp allele retained its prominence over the ensuing period, the 791-bp product declined in intensity. Nonetheless, it was still detectable in the day 18 sample.

Genotypic analysis of breakthrough parasites following transmission. To enable an examination of the genotype structure of the breakthrough parasite population, nymphal R. appendiculatus ticks were applied to the animal on days 9, 10, 12, and 13 after challenge. Engorged ticks detaching on days 17 and 18 were collected and used to generate a working sporozoite stabilate (St105) as described above. Individual parasitized cell lines derived by in vitro infection and cloning were then examined using the panel of 64 polymorphic markers. A total of 89 clones were examined, 74 of which were attributable to a single genotype, designated 105-01. An additional genotype (105-02) accounted for eight of the clones. Two other genotypes were represented by pairs, while three were present as singletons. No occurrence of the 72-01 genotype used for immunization was observed. The dominant 105-01 genotype was the most distantly related to that of 72-01, sharing only 35 (55%) of its alleles. The 105-02 genotype was more closely related, sharing 52 (81%) of the 72-01 alleles, while the remaining genotypes were intermediate in their relatedness. A comparison of the frequencies of 72-01 alleles at individual marker loci across all four chromosomes in St72 and the post-immune selection St105 population is shown in Fig. 4. Although a number of loci showed a substantial reduction in the frequency of 72-01 alleles following passage through the immune calf, only one locus, TP01_0966, was entirely devoid of the allele present in 72-01. A marked decline in 72-01 allele frequencies was evident in segments of each of the four chromosomes, as delineated by contiguous markers. The largest of these segments was on chromosome 4 and spanned approximately 420 kb. In the light of our previous observations of a considerable increase in the frequency of 72-01 alleles in a stabilate derived from ticks fed on a naïve calf infected with St72, these observations reflect a substantial effect of the immune response on the transmission of alleles associated with the immunizing clone.

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FIG. 4. Frequencies of 72-01 alleles in clones derived from St72 (•; n = 231) and St105 (; n = 89), at individual marker loci arrayed sequentially across the T. parva genome. The regions corresponding to individual chromosomes are indicated.

An additional experiment was conducted to determine whether immune responses engendered by the 72-01 genotype in animals with distinct MHC phenotypes result in different breakthrough populations. Five unrelated male Friesian calves aged 4 months were selected by typing PBMC with a panel of discriminatory MHC class I-specific monoclonal antibodies (data not shown). The animals were immunized against the 72-01 genotype by inoculation with autologous cells infected with the clone as described above and challenged 94 days later with 1.25 tick equivalents of the bulk stabilate St72. None of the animals developed clinical disease following challenge, although transient fever was observed, along with evidence of lymph node hyperplasia. Lymph node aspirates and blood samples collected at periodic intervals after challenge were used to prepare DNA as described above and were analyzed with selected markers. The results of such an analysis with the TP04_0051 marker, which showed the greatest sensitivity and discrimination, are shown in Fig. 5. In contrast with observations in animal 211, the 791-bp allele characteristic of the 72-01 genotype was absent from lymph node aspirates of all but two of the calves and was detectable in the erythrocyte fraction of only one of these animals. However, each calf exhibited a distinct profile with respect to the emergence of other parasite genotypes in terms of both the alleles present and their relative abundances (Fig. 5). Similar observations were made with the other markers evaluated (data not shown). Hence, the impact of the immune response on the genotypic structure of the challenge population appears to vary between animals.

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FIG. 5. Genotype analysis of breakthrough parasites in five unrelated calves immunized against the 72-01 T. parva genotype and challenged with the parent stabilate, St72. The profile for the TP04_0051 polymorphic marker is shown. The lanes contain samples derived from lymph node (L) or erythrocyte (E) compartments on the indicated days after challenge. The arrowheads indicate the bands representing the allele present in the 72-01 genotype.

Influence of the immune response on parasite transmission. A major question regarding the ability of bovine CTL immunity to impose selection on T. parva is whether it prevents or constrains differentiation of the parasite to transmissible piroplasm forms. We addressed this question using two immunologically cross-reactive but molecularly distinct populations of T. parva. Six male Friesian calves aged 4 months were immunized with St71 of the Muguga isolate as outlined above and challenged, along with two naïve control animals, approximately 8 months later by subcutaneous inoculation with 20 tick equivalents of the monotypic 72-01 stabilate St103. Immunization with St71 confers protection against 72-01 parasites, and the stabilate is near clonal (data not shown). The two populations differ at 15 of the 64 marker loci, allowing unequivocal identification of parasite genotypes in the piroplasm population. Evidence for cytolytic activity against both parasite populations was observed prechallenge in PBMC from five of the six animals after in vitro restimulation with autologous cells infected with the Muguga isolate of T. parva; data were unavailable from the remaining calf because it was not possible to establish a 72-01-infected line from it (data not shown). Lymph node aspirates and blood samples were collected on alternate days from day 6 after challenge and used to prepare DNA as described above for analysis with selected markers. All six immunized animals underwent mild self-limiting infections after challenge with St103, while the two naïve control calves developed severe clinical disease. Schizont forms of the parasite were detected in lymph node aspirates in all cases, although piroplasms were observed in only two of the immunized calves on single occasions. However, when RBC fractions of blood sampled from the calves following challenge were examined for the presence of parasites by PCR using the MS49 minisatellite marker (13), evidence of piroplasms was obtained in all cases (Fig. 6). In each instance, the allele present was that of 72-01 rather than the immunizing Muguga parasite, indicating that parasite challenge can give rise to transmissible forms in spite of a specific CTL response and effective immunity.

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FIG. 6. Genotypic analysis of T. parva parasites in the erythrocyte compartments of six calves challenged, along with two naïve control animals, with the 72-01 stabilate St103 approximately 8 months after immunization with the Muguga isolate of the parasite. The profile for the discriminatory marker MS49 is shown for samples obtained on day 17 after challenge. Profiles of the immunizing and challenge populations are presented on the left for comparison.

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We have previously demonstrated substantial genotypic diversity within the Marikebuni isolate of T. parva and provided evidence for frequent meiotic recombination of the parasite in the tick vector (13). Such diversity clearly has implications for immune recognition and raises questions regarding the extent to which protective immune responses in cattle modulate the population structure of the parasite. We now extend our previous observations to characterize the influence exerted by the immune response on the diversity of parasites transmitted by infected cattle.

Our previous work using the CTVM St72 stabilate of T. parva revealed that the parasite population that emerges following challenge of a naïve calf broadly mirrors that present in the stabilate and is dominated by the 72-01 genotype. Furthermore, transmission of this population through the tick vector retains the dominance of the 72-01 genotype and increases the frequencies of its alleles among the residual genotypes. We now provide evidence that prior immunization with a cloned parasite carrying the 72-01 genotype results in substantial modulation of the parasite population that emerges following challenge with the bulk stabilate. First, no instances of the immunizing genotype were observed in cloned parasitized cell lines derived from lymph node biopsy specimens taken on three occasions during the course of infection. Second, analysis of whole-biopsy DNA with the TP04_0051 marker at several time points during the patent infection revealed a rapid decline in the prevalence of the 72-01 allele accompanied by the emergence of two distinct alleles. These observations provide clear evidence that the immune response effectively curtails expansion of the immunizing genotype from the challenge population. The strong body of evidence that control of the schizont parasitosis is CD8⁺ T-cell dependent supports the conclusion that this effect arises from specific CTL harbored by the calf prior to challenge.

A decline in the prevalence of the 72-01 allele of the TP04_0051 marker was also observed in the erythrocyte fraction during the course of challenge, indicating that the impact of the CTL response on the expansion of the immunizing genotype is reflected in significant reduction of its prevalence in the piroplasm population. This was confirmed across the genome by transmission of the piroplasm complement through the tick vector. Analysis of the prevalence of 72-01 alleles among 89 parasite clones derived from R. appendiculatus ticks fed on the calf revealed greater than 70% reduction in the frequency of alleles at 28 of 64 loci tested (Fig. 4). Only one locus, which encodes TP01_0966, a hypothetical protein potentially located in the apicoplast (4), was completely devoid of the 72-01 allele. It might be speculated that this locus constituted a marker for a major antigenic target of the response in this animal, although the precision of such a prediction would be limited, given that the adjacent markers flank a stretch of 600 kb. Martinelli and others (14) observed so-called "selection valleys" in an analysis of the impact of strain-specific immunity across the genome in the Plasmodium chabaudi chabaudi mouse model of malaria. On this basis, they identified the merozoite surface protein 1 as a major target of selection. However, the analogies between their system and that examined in the current study are somewhat limited. In particular, their conclusions were based on direct comparison of parasites emerging in immune and naïve mice, whereas we compared parasites before and after transmission through an immune animal. Given our observation that even parasites targeted by the immune response progress to transmissible forms, the predictive power of this approach for antigen identification may be somewhat limited. In this regard, the "valleys" apparent in the genome-wide frequencies of 72-01 alleles in the St105 population (Fig. 4) do not correspond to those observed in a similar analysis of clones derived from lymph node biopsy specimens taken during the course of the breakthrough infection (data not shown).

These analyses provide clear evidence for a substantive reduction in the prevalence of the immunizing genotype following challenge of an immune calf with the bulk stabilate. However, since many of the remaining components of the stabilate share a significant proportion of marker alleles with the 72-01 genotype, it is difficult to estimate the real impact of the CTL response on transmission of targeted parasites in this experimental system. Hence, it is unclear whether piroplasms carrying 72-01 alleles in the breakthrough population arose from 72-01 itself or from other components of the challenge infection. Some clarity in this regard emerges from an additional experiment in which six calves were challenged with the 72-01 clone 8 months after immunization with the genotypically distinct but cross-reacting Muguga isolate of T. parva. Although all of the calves showed a high level of protection, piroplasms arising from the challenge parasite were detected in each animal, as evidenced by the appearance of the distinct marker allele in the erythrocyte fraction. This observation provides compelling evidence that bovine CTL response against T. parva reduces but fails to abrogate transmission in immune cattle under challenge. The absence of the Muguga allele in material from the immunized calves at the time of challenge is noteworthy and is consistent with previous reports that this isolate gives rise to only a transient carrier state (23, 29).

Recent findings have indicated that the antigenic specificity of the CTL response against T. parva varies between animals of different MHC phenotypes (9), confirming previous observations of immunodominance and strain specificity (7, 8, 30). The observation that different parasite genotype profiles were found in the erythrocyte fractions of five unrelated 72-01-immune calves following challenge with St72 suggests that such variation in the specificity of the response is reflected in modulation of parasite transmission at the population level. The qualitative impact of the CTL response on parasite transmission therefore appears to be variable between animals. Given that qualitative constraints imposed by parasite-specific CTL on transmission are dictated by the specificity of the response, which in turn is dependent on the host MHC phenotype, the selective power of the response on the parasite at a population level would be expected to depend on the level of heterogeneity among herd MHC haplotypes. Hence, repeated field transmission of a given parasite population through cattle with low MHC heterogeneity and similar CTL specificities, for example, in more intensively managed herds utilizing artificial insemination and selecting for production traits, would result in continual selection against targeted loci and favor diversity in the antigens in question. On the other hand, high levels of herd MHC diversity, as might be expected in more extensive systems, would dilute selection on individual loci and hence reduce antigenic polymorphism in resident parasite populations. Clarification of these issues will await an evaluation of antigenic diversity in the parasites prevailing under each of these circumstances.

A striking feature of the St72 population is the dominance of the 72-01 genotype, which accounts for over 75% of its constituent parasites. The reasons for this are unclear, although we have proposed that it may have arisen from selective processes in the tick vector (13). The emergence of a distinct genotype as a dominating population in ticks fed on the 72-01-immune calf after challenge with St72 perhaps challenges that suggestion, given that the same tick line was used for the production of both stabilates and would be expected to continue to favor 72-01 alleles. Indeed, the most dominant of the seven genotypes identified in St105 was that which shared the fewest alleles with 72-01. This argues against selection in the tick as a sole driver for the emergence of dominant genotypes and suggests that host and/or parasite factors may also play a role.

In conclusion, we have observed that protective immunity against T. parva has considerable impact on the emergence of targeted parasites following challenge but fails to prevent their differentiation into transmissible forms. Furthermore, the genotypic compositions of the transmitted parasites arising from a given challenge vary between immune individuals with distinct MHC phenotypes. This suggests that the intensity of selection in settings where the parasite is endemic may be influenced by the extent of herd MHC heterogeneity. These observations almost certainly have relevance for other vector-borne apicomplexans, including the closely related T. annulata and Plasmodium species.

 
We are grateful to Niall MacHugh and Ivan Morrison for critical review of the manuscript and to Hazel Simm for formatting the figures.

The work described was wholly funded by the Wellcome Trust (project grants 064654 and 077431).

 
* Corresponding author. Present address: Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom. Phone: 44 1707 666318. Fax: 44 1707 666402. E-mail: dmckeever{at}rvc.ac.uk

Published ahead of print on 16 July 2007.

Editor: W. A. Petri, Jr.

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Infection and Immunity, October 2007, p. 4909-4916, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00710-07
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