From BLAST analyses, the genome of Cryptosporidium, like that of Plasmodium falciparum 26, is more similar overall to those of the plants Arabidopsis and Oryza than to any other non-apicomplexan organism currently represented in GenBank. The program Glimmer predicted 5,519 protein-coding sequences in the C. parvum genome, 4,320 of which had similarity to other sequences deposited in the GenBank nonredundant protein database. A significant number of these sequences, 936 (E-value < 10^-3) or 783 (E-value < 10^-7), had their most significant, non-apicomplexan, similarity to a sequence isolated from plants, algae, eubacteria (including cyanobacteria) or archaea (Table 1). To evaluate these observations further, phylogenetic analyses were performed, when possible, for each predicted protein in the entire genome.

The Glimmer-predicted protein-coding regions of the C. parvum genome (5,519 sequences) were used as input for phylogenetic analyses using the PyPhy program 27. In this program, phylogenetic trees for each input sequence are analyzed to determine the taxonomic identity of the nearest neighbor relative to the input sequence at a variety of taxonomic levels, for example, genus, family, or phylum. Using stringent analysis criteria (see Materials and methods), 954 trees were constructed from the input set of 5,519 predicted protein sequences (Figure 1). Analysis of the nearest non-apicomplexan neighbor on the 954 trees revealed the following nearest neighbor relationships: eubacterial (115 trees), archaeal (30), green plant/algal (204), red algal (8), and glaucocystophyte (4); other alveolate (61) and other eukaryotes made up the remainder. As some input sequences may have more than one nearest neighbor of interest on a tree, a nonredundant total of 393 sequences were identified with nearest neighbors to the above lineages.

Searches of the C. parvum predicted gene set with the 551 P. falciparum predicted nuclear-encoded apicoplast-targeted proteins (NEAPs) yielded 40 significant hits (E-value < 10^-5), 23 of which were also identified in the phylogenomic analyses. A combination of these two approaches identified 410 candidates requiring further detailed analyses. Of these candidates, the majority were eliminated after stringent criteria were applied because of ambiguous tree topologies, insufficient taxonomic sampling, lack of bootstrap support or the presence of clear vertical eukaryotic ancestry (see Materials and methods). Thirty-one genes survived the screen and were deemed to be either strong or likely candidates for gene transfer (Table 2).

Of the 31 recovered genes, several have been previously published or submitted to the GenBank 20, including those identified as having plant or eubacterial 'likeness' on the basis of similarity searches when the genome sequence was published 7. The remaining sequences were further tested to rule out the possibility that they were artifacts (C. parvum oocysts are purified from cow feces which contain plant and bacterial matter). Two experiments were performed. In the first, nearly complete genomic sequences (generated in a different laboratory) from the closely related species C. hominis were screened using BLASTN for the existence of the predicted genes. Twenty out of 21 C. parvum sequences were identified in C. hominis. The remaining sequence was represented by two independently isolated expressed sequence tag (EST) sequences in the GenBank and CryptoDB databases (data not shown). In the second experiment, genomic Southern analyses of the IOWA isolate were carried out (Figure 2) for several of the genes of bacterial or plant origin. In each case, a band of the predicted size was identified (see Additional data file 1). The genes are not contaminants.

Extant Cryptosporidium species do not contain an apicoplast genome or any physical structure thought to represent an algal endosymbiont or the plastid organelle it contained 67. The only possible remaining evidence of the past association of an endosymbiont or its cyanobacterially derived plastid organelle might be genes transferred from these genetic sources to the host genome prior to the physical loss of the endosymbiont or organelle itself. Several such genes were identified.

A leucine aminopeptidase gene of cyanobacterial origin was found in the C. parvum nuclear genome. This gene is also present in the nuclear genome of other apicomplexan species (Plasmodium, Toxoplasma and Eimeria), as confirmed by similarity searches against ApiDB (see Materials and methods). In P. falciparum, leucine aminopeptidase is a predicted NEAP and possesses an amino-terminal extension with a putative transit peptide. Consistent with the lack of an apicoplast, this gene in Cryptosporidium contains no evidence of a signal peptide and the amino-terminal extension is reduced. Similarity searches of the GenBank nonredundant protein database revealed top hits to Plasmodium, followed by Arabidopsis thaliana, and several cyanobacteria including Prochlorococcus, Nostoc and Trichodesmium, and plant chloroplast precursors in Lycopersicon esculentum and Solanum tuberosum (data not shown). A multiple sequence alignment of the predicted protein sequences of leucine aminopeptidase reveals overall similarity and a shared indel among apicomplexan, plant and cyanobacterial sequences (Figure 3). Phylogenetic analyses strongly support a monophyletic grouping of C. parvum and other apicomplexan leucine aminopeptidase proteins with cyanobacteria and plant chloroplast precursors (Figure 4a). So far, this gene has not been detected in ciliates.

Another C. parvum nuclear-encoded gene of putative cyanobacterial origin is a protein of unknown function belonging to the biopterine transporter family (BT-1) (Table 2). Similarity searches with this protein revealed significant hits to other apicomplexans (for example, P. falciparum, Theileria annulata, T. gondii), plants (Arabidopsis, Oryza), cyanobacteria (Trichodesmium, Nostoc and Synechocystis), a ciliate (Tetrahymena) and the kinetoplastids (Leishmania and Trypanosoma). Arabidopsis thaliana apparently contains at least two copies of this gene; the protein of one (accession number NP_565734) is predicted by ChloroP 28 to be chloroplast-targeted, suggestive of its plastid derivation. The taxonomic distribution and sequence similarity of this protein with cyanobacterial and chloroplast homologs are also indicative of its affinity to plastids.

Only one gene of algal nuclear origin, glucose-6-phosphate isomerase (G6PI), was identified by the screen described here. Several other algal-like genes are probable, but their support was weaker (Table 2). A 'plant-like' G6PI has been described in other apicomplexan species (P. falciparum, T. gondii 29) and a 'cyanobacterial-like' G6PI has been described in the diplomonads Giardia intestinalis and Spironucleus and the parabasalid Trichomonas vaginalis 30. Figure 4b illustrates these observations nicely. At the base of the tree, the eukaryotic organisms Giardia, Spironucleus and Trichomonas group with the cyanobacterium Nostoc, as previously published. In the midsection of the tree, the G6PI of apicomplexans and ciliates forms a well-supported monophyletic group with the plants and the heterokont Phytophthora. The multiple protein sequence alignment of G6PI identifies several conserved positions shared exclusively by apicomplexans, Tetrahymena, plants and Phytophthora. This gene does not contain a signal or transit peptide and is not predicted to be targeted to the apicoplast in P. falciparum. The remainder of the tree shows a weakly supported branch including eubacteria, fungi and several eukaryotes. The eukaryotes are interrupted by the inclusion of G6PI from the eubacterial organisms Escherichia coli and Cytophaga. This relationship of E. coli G6PI and eukaryotic G6PI has been observed before and may represent yet another gene transfer 31.

Our study identified HGTs from several distinct sources, involving a variety of biochemical activities and metabolic pathways (Table 2). Notably, the nucleotide biosynthesis pathway contains at least two previously published, independently transferred genes from eubacteria. Inosine 5' monophosphate dehydrogenase (IMPDH), an enzyme for purine salvage, was transferred from ε-proteobacteria 32. Another enzyme involved in pyrimidine salvage, thymidine kinase (TK), is of α or γ-proteobacterial ancestry 25.

Another gene of eubacterial origin identified in C. parvum is tryptophan synthetase β subunit (trpB). This gene has been identified in both C. parvum and C. hominis, but not in other apicomplexans. The relationship of C. parvum trpB to proteobacterial sequences is well-supported as a monophyletic group by two of the three methods used in our analyses (Figure 4c).

Other HGTs of eubacterial origin include the genes encoding α-amylase and glutamine synthetase and two copies of 1,4-α-glucan branching enzyme, all of which are overwhelmingly similar to eubacterial sequences. α-amylase shows no significant hit to any other apicomplexan or eukaryotic sequence, suggesting a unique HGT from eubacteria to C. parvum. Glutamine synthetase is a eubacterial gene found in C. parvum and all apicomplexans examined. The eubacterial affinity of the apicomplexan glutamine synthetase is also demonstrated by a well supported (80% with maximum parsimony) monophyletic grouping with eubacterial homologs (data not shown). The eubacterial origin of 1,4-α-glucan branching enzyme is shown in Figure 5. Each copy of the gene is found in a strongly supported monophyletic group of sequences derived only from prokaryotes (including cyanobacteria) and one other apicomplexan organism, T. gondii. It is possible that these genes are of plastidic origin and were transferred to the nuclear genome before the divergence of C. parvum and T. gondii; the phylogenetic analysis provides little direct support for this interpretation, however.

We examined the transferred genes for evidence of non-independent acquisition, for example, blocks of transferred genes or evidence that genes were acquired together from the same source. Examination of the chromosomal location of the genes listed in Table 2 demonstrates that the genes are currently located on different chromosomes and in most cases do not appear to have been transferred or retained in large blocks. There are two exceptions. The trpB gene and the gene for aspartate ammonia ligase are located 4,881 base-pairs (bp) apart on the same strand of a contig for chromosome V; there is no annotated gene between these two genes. Both genes are of eubacterial origin and are not found in other apicomplexan organisms. While it is possible that they have been acquired independently with this positioning, or later came to have this positioning via genome rearrangements, it is interesting to speculate that these genes were acquired together. The origin of trpB is proteobacterial. The origin of aspartate ammonia ligase is eubacterial, but not definitively of any particular lineage. In the absence of genome sequences for all organisms, throughout all of time, exact donors are extremely difficult to assess and inferences must be drawn from sequences that appear to be closely related to the actual donor.

In the second case, C. parvum encodes two genes for 1,4-α-glucan branching enzymes. Both are eubacterial in origin and both are located on chromosome VI, although not close together. They are approximately 110 kb apart and many intervening genes are present. The evidence that these genes were acquired together comes from the phylogenetic analysis presented in Figure 5. The duplication that gave rise to the two 1,4-α-glucan branching enzymes is old, and is well supported by the tree shown in Figure 5. A number of eubacteria (11), including cyanobacteria, contain this duplication. The 1,4-α-glucan branching enzymes of C. parvum and T. gondii represent one copy each of this ancient duplication. This suggests that the ancestor of C. parvum and T. gondii acquired the genes after they had duplicated and diverged in eubacteria.

Each of the genes identified in the above analyses (Table 2) appears to be an intact non-pseudogene, suggesting that these genes are functional. To verify the functional status of several of the transferred genes, semi-quantitative reverse transcription PCR (RT-PCR) was carried out to characterize their developmental expression profile. Each of the RNA samples from C. parvum-infected HCT-8 cells was shown to be free of contaminating C. parvum genomic DNA by the lack of amplification product from a reverse transcriptase reaction sham control. RT-PCR detected no signals in cDNA samples from mock-infected HCT-8 cells. On the other hand, RT-PCR product signals were detected in the C. parvum-infected cells of six independent time-course experiments for each of the genes examined (those for G6PI, leucine aminopeptidase, BT-1, a calcium-dependent protein kinase, tyrosyl-tRNA synthetase, dihydrofolate reductase- thymidine synthetase (DHFR-TS)). The expression profiles of the acquired genes show that they are regulated and differentially expressed throughout the life cycle of C. parvum in patterns characteristic of other non-transferred genes (Figure 6).

A small published collection of 567 EST sequences for C. parvum is also available. These ESTs were searched with each of the 31 candidate genes surviving the phylogenomic screen. Three genes - aspartate ammonia ligase, BT-1 and lactate dehydrogenase - are expressed, as confirmed by the presence of an EST (Table 2).

The C. parvum sequences of cyanobacterial and algal origin reported here had to enter the genome at some point during its evolution. Formal possibilities include vertical inheritance from a plastid-containing chromalveolate ancestor, HGT from the cyanobacterial and algal sources (or from a secondary source such as a plastid-containing apicomplexan), or IGT from an endosymbiont/plastid organelle during evolution, followed by loss of the source. Cryptosporidium does not harbor an apicoplast organelle or any trace of a plastid genome 7; thus an IGT scenario would necessitate loss of the organelle in Cryptosporidium or the lineage giving rise to it. The exact position of C. parvum on the tree of life has been debated, with developmental and morphological considerations placing it within the Apicomplexa, and molecular analyses locating it in various positions, both within and outside the Apicomplexa 3, but primarily within. If we assume that C. parvum is an apicomplexan, and if the secondary endosymbiosis which is believed to have given rise to the apicoplast occurred before the formation of the Apicomplexa, as has been suggested 18, C. parvum would have evolved from a plastid-containing lineage and would be expected to harbor traces of this relationship in its nuclear genome. Genes of likely cyanobacterial and algal/plant origin are detected in the nuclear genome of C. parvum (Table 2) and thus IGT followed by organelle loss cannot be ruled out.

What about other interpretations? While it is formally possible that these genes were acquired independently via HGT in C. parvum, their shared presence in other alveolates (including the non-plastidic ciliate Tetrahymena) provides the best evidence against this scenario as multiple independent transfers would be required and so far there is no evidence for intra-alveolate gene transfer. Vertical inheritance is more difficult to address as it involves distinguishing between genes acquired via IGT from a primary endosymbiotic event versus a secondary endosymbioic event. Our data, especially the analysis of G6PI and BT-1 are consistent with both primary and secondary endosymbioses, provided that the secondary endosymbiosis is pre-alveolate in origin. As more genome data become available and flanking genes can be examined for each gene in a larger context, positional information will be informative in distinguishing among the alternatives.

The plastidic nature of some genes is particularly apparent. There is a shared indel among leucine aminopeptidase protein sequences in apicomplexans, cyanobacteria and plant chloroplast precursors (Figure 3). The C. parvum leucine aminopeptidase does contain an amino-terminal extension of approximately 85-65 amino acids (depending on the alignment) relative to bacterial homologs, but this extension does not contain a signal sequence. The extension in P. falciparum is 85 amino acids and the protein is believed to be targeted to the apicoplast 2637. No similarity is detected between the C. parvum and P. falciparum amino-terminal extensions (data not shown).

Other genes were less informative in this analysis. Among these, aldolase was reported in both P. falciparum 38 and the kinetoplastid parasite Trypanosoma 38 as a plant-like gene. The protein sequences of aldolase are similar in C. parvum and P. falciparum, with an identity of 60%. In our phylogenetic analyses, C. parvum clearly forms a monophyletic group with Plasmodium, Toxoplasma and Eimeria. This branch groups with Dictyostelium, Kinetoplastida and cyanobacterial lineages, but bootstrap support is not significant. The sister group to the above organisms are the plants and additional cyanobacteria, but again with no bootstrap support (see Additional data file 1 for phylogenetic tree). Another gene, enolase, contains two indels shared between land plants and apicomplexans (including C. parvum) and was suggested to be a plant-like gene 29, but alternative explanations exist 39.

The biochemical activity of the polyamine biosynthetic enzyme arginine decarboxylase (ADC), which is typically found in plants and bacteria, was previously reported in C. parvum 19. However, we were unable to confirm its presence by similarity searches of the two Cryptosporidium genome sequences deposited in CryptoDB using plant (Cucumis sativa, GenBank accession number AAP36992), cyanobacterial (Nostoc sp., NP-487441; Synechocystis sp., NP-439907) and other bacterial (Yersinia pestis, NP-404547) homologs.

Several HGTs from bacteria have been reported previously in C. parvum 253240. We detected many more in our screen of the completed C. parvum genome sequence (Table 2). In most cases, the exact donors of these transferred genes were difficult to determine. However, for those genes whose donors could be more reliably inferred (Table 2), several appear to be from different sources and hence represent independent transfer events. In one compelling case, both the trpB and aspartate ammonia ligase genes are located 4,881 bp apart on the same strand of a contig for chromosome V and there is no gene separating them. Both genes are of eubacterial origin and neither gene is detected in other apicomplexans. In addition, the aspartate ammonia ligase gene is expressed, as evidenced by an EST. In another case, copies of a 1,4-α-glucan branching enzyme gene duplication pair that is present in many eubacteria, were detected on the same chromosome in C. parvum. C. parvum also contains many transferred genes from distinct eubacterial sources that are not present in other apicomplexans (for example, IMPDH, TK (thymidine kinase), trpB and the gene for aspartate ammonia ligase).

The endosymbiotic event that gave rise to the mitochondrion occurred very early in eukaryotic evolution and is associated with significant IGT. However, most of these transfer events happened long before the evolutionary time window we explored in this study 41. Many IGTs from the mitochondrial genome that have been retained are almost universally present in eukaryotes (including C. parvum which does not contain a typical mitochondrion 7424344) and thus would not be detected in a PyPhy screen since the 'nearest phylogenetic neighbor' on the tree would be taxonomically correct and not appear as a relationship indicative of a gene transfer.

Gene transfer is an important evolutionary force 21224546. Several of the transferred genes identified in C. parvum are known to be expressed. IMPDH has been shown to be essential in C. parvum purine metabolism 32 and TK has been shown to be functional in pyrimidine salvage 25. It is not yet clear whether these genes were acquired independently in this lineage, or have been lost from the rest of the apicomplexan lineage, or whether both these have happened. However, it is clear that their presence has facilitated the remodeling of nucleotide biosynthesis. C. parvum no longer possesses the ability to synthesize nucleotides; instead it relies entirely on salvage.

Many apicoplast and algal nuclear genes have been transferred to the host nuclear genome, where they were subsequently translated in the cytosol and their proteins targeted to the apicoplast organelle. However, as there is no apicoplast in C. parvum, acquired plastidic proteins are theoretically destined to go elsewhere. In the absence of an apicoplast, it is tempting to suspect that plastid-targeted proteins would have been lost, or would be detected as pseudogenes. No identifiable pseudogenes were detected and at least one gene is still viable. The C. parvum leucine aminopeptidase, which still contains an amino-terminal extension (without a signal peptide), is intact and is expressed, as shown in Figure 6. None of the cyanobacterial/algal genes identified in our study contains a canonical presequence for apicoplast targeting. One exception to this is phosphoglucomutase, a gene not present in Table 2 because of its poorly supported relationships in phylogenetic analyses. This gene exists in two copies as a tandem duplication in the C. parvum genome. One copy has a long amino-terminal extension (97 amino acids) beginning with a signal peptide. The extension does not contain characteristics of a transit peptide. Expression of a fluorescent reporter construct containing this extension in a related parasite, T. gondii, did not reveal apicoplast targeting but instead secretion via dense granules (see Additional data file 1). Exactly how and where intracellularly transferred genes (especially those that normally target the apicoplast) have become incorporated into other metabolic processes remains a fertile area for exploration.

Genomic sequences for C. parvum and C. hominis were downloaded from CryptoDB 53. Genes were predicted for the completed C. parvum (IOWA) sequence as previously described using the Glimmer program 54 trained on Cryptosporidium coding sequences 52. A few predicted genes that demonstrated apparent sequence incompleteness were reconstructed from genomic sequence by comparison with apicomplexan orthologs. The predicted protein encoding data set contained 5,519 sequences. A comparison of this gene set to the published annotation revealed that the Glimmer-predicted gene set contained all but 40 of the 3,396 annotated protein-encoding sequences deposited in GenBank. These 40 were added to our dataset and analyzed. Glimmer does not predict introns and some introns are present in the genome 720; thus our gene count is artificially inflated. Likewise, the official C. parvum annotation did not consider ORFs of less than 100 amino acids that did not have significant BLAST hits and thus may be a slight underestimate 7.

An internal database (ApiDB) containing all available apicomplexan sequence data was created 25. A second BLAST-searchable database, PyPhynr, was constructed that included SwissProt, TrEMBL and TrEMBL_new, as released in August 2003, predicted genes from C. parvum, ORFs of more than 120 amino acids from Theileria annulata, and more than 75 amino acids from consensus ESTs for several apicomplexan organisms. Genomic sequences for T. gondii (8x coverage) and clustered ESTs were downloaded from ToxoDB 5556. Genomic data were provided by The Institute for Genomic Research (TIGR), and by the Sanger Institute. EST sequences were generated by Washington University. In addition, this study used sequence data from several general and species-specific databases. Specifically, the NCBI GenBank nr and dbEST were downloaded 57 and extensively searched. To provide taxonomic completeness, additional genes were obtained via searches of additional databases including: Entamoeba histolytica 58, D. discoideum 59, the kinetoplastids Leishmania major 59, T. brucei 59, T. cruzi 60, and a ciliate Tetrahymena thermophila 61. Sequence data for T. annulata, E. histolytica, D. discoideum, L. major and T. brucei were produced by the Pathogen Sequencing Unit of the Sanger Institute and can be obtained from 62. Preliminary sequence data for T. thermophila was obtained from TIGR and can be accessed at 63.

The source code of the phylogenomic software PyPhy 27 was kindly provided by Thomas Sicheritz-Ponten and modified to include analyses of eukaryotic groups, and changes to improve functionality 51. For initial phylogenomic analyses, a BLAST cutoff of 60% sequence length coverage and 50% sequence similarity was adopted and the neighbor-joining program of PAUP 4.0b10 for Unix 64 was used. A detailed description of our phylogenomic pipeline and PyPhy implementation are described 51 and outlined in Figure 1.

Output gene trees with phylogenetic connections (that is, the nearest non-self neighbors at a distinct taxonomic rank) 27 to prokaryotes and algae-related groups were manually inspected. As the trees are unrooted, several factors were considered in the screen for candidate transferred genes. If the C. parvum gene does not form a monophyletic group with prokaryotic or plant-related taxa regardless of rooting, the subject gene was eliminated from further consideration. If the topology of the gene tree is consistent with a phylogenetic anomaly caused by gene transfer, but may also be interpreted differently if the tree is rooted otherwise, it was removed from consideration at this time. If the top hits of both nr and dbEST database searches are predominantly non-plant eukaryotes, and the topology of the tree was poor, the subject gene was considered an unlikely candidate. Finally, all 551 protein sequences predicted to be NEAPs in the malarial parasite P. falciparum 26 were used to search the C. parvum genome and the results were screened using a BLAST cutoff E-value of 10^-5 and a length coverage of 50%. Sequences identified by these searches were added to the candidate list (if not already present) for manual phylogenetic analyses to verify their likely origins. It should be noted that all trees were screened for the existence of a particular phylogenetic relationship. In some cases the proteins utilized to generate a particular tree are capable of resolving relationships among many branches of the tree of life, and in others they are not. Despite these differences in resolving power, the proteins which survive our phylogenetic screen and subsequent detailed analyses described below exhibit significant support for the branches of the tree in which we are interested. Similar procedures were used to characterize the complement of nuclear-encoded genes of plastid origin in the Arabidopsis genome 65. BLAST searches were performed on GenBank releases 138-140 57.

Detailed phylogenetic analyses of candidate genes identified by phylogenomic screening: candidate genes surviving the PyPhy phylogenomic screen were reanalyzed with careful attention to taxonomic completeness, including representative species from major prokaryotic and eukaryotic lineages when necessary and possible. New multiple sequence alignments were created with ClustalX 66, followed by manual refinement. Only unambiguously aligned sequence segments were used for subsequent analyses (see Additional data file 1). Phylogenetic analyses were performed with a maximum likelihood method using TREE-PUZZLE version 5.1 for Unix 67, a distance method using the program neighbor of PHYLIP version 3.6a package 68, and a maximum parsimony method with random stepwise addition using PAUP* 4.0b10 64. Bootstrap support was estimated using 1,000 replicates for both parsimony and distance analyses and quartet puzzling values were obtained using 10,000 puzzling steps for maximum likelihood analyses. Distance calculation used the Jones-Taylor-Thornton (JTT) substitution matrix 69, and site-substitution variation was modeled with a gamma-distribution whose shape parameter was estimated from the data. For maximum likelihood analyses, a mixed model of eight gamma-distributed rates and one invariable rate was used to calculate the pairwise maximum likelihood distances. The unrooted trees presented in Figures 4 and 5 were drawn by supplying TREE-PUZZLE with the maximum parsimony tree and using TREE-PUZZLE distances as described above to calculate the branch lengths. The trees were visualized and prepared for publication with TreeView X Version 0.4.1 70.

C. parvum (IOWA) oocysts (10⁸) were obtained from the Sterling Parasitology Laboratory at the University of Arizona and were lysed using a freeze/thaw method. Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen). Genomic DNA (5 μg) was restricted with BamH1 and EcoR1 respectively and electrophoresed on a 0.8% gel in 1x TAE buffer, transferred to a positively charged nylon membrane (Bio-Rad), and fixed using a UVP crosslinker set at 125 mJ as described in 71. C. parvum genomic DNA for the probes (700-1,500 bp) was amplified by PCR (see Additional data file 1).

Sterilized C. parvum (IOWA isolate) oocysts were used to infect confluent human adenocarcinoma cell monolayers at a concentration of one oocyst per cell as previously described 72. Total RNA was prepared from mock-infected and C. parvum-infected HCT-8 cultures at 2, 6, 12, 24, 36, 48 and 72 h post-inoculation by directly lysing the cells with 4 ml TRIzol reagent (GIBCO-BRL/Life Technologies). Purified RNA was resuspended in RNAse-free water and the integrity of the samples was confirmed by gel electrophoresis.

Primers specific for several transferred genes identified in the study were designed (see Additional data file 1) and a semi-quantitative RT-PCR analysis was carried out as previously described 72. Primers specific for C. parvum 18S rRNA were used to normalize the amount of cDNA product of the candidate gene to that of C. parvum rRNA in the same sample. PCR products were separated on a 4% non-denaturing polyacrylamide gel and signals from specific products were captured and quantified using a phosphorimaging system (Molecular Dynamics). The expression level of each gene at each time point was calculated as the ratio of its RT-PCR product signal to that of the C. parvum 18S rRNA. Six independent time-course experiments were used in the analysis.