INTRODUCTION
Hammondia heydorni, Hammondia hammondi, Toxoplasma gondii, Neospora hughesi and Neospora caninum are closely related tissue cyst-forming coccidians with a 2-host life-cycle. Hammondia heydorni and N. caninum use canids as definitive hosts whereas felids are the definitive hosts for T. gondii and H. hammondi (Frenkel and Dubey, Reference Frenkel and Dubey1975; McAllister et al. Reference McAllister, Dubey, Lindsay, Jolley, Wills and McGuire1998; Lindsay et al. Reference Lindsay, Dubey and Duncan1999, Reference Lindsay, Ritter and Brake2001; Gondim et al. Reference Gondim, McAllister, Pitt and Zemlicka2004). Because the oocyst stage of these coccidians is morphologically similar, fecal diagnosis is difficult and there is intense debate about their taxonomy and phylogenetic relationships (Ellis et al. Reference Ellis, Morrison, Liddell, Jenkins, Mohammed, Ryce and Dubey1999; Dubey et al. Reference Dubey, Barr, Barta, Bjerkas, Björkman, Blagburn, Bowman, Buxton, Ellis, Gottstein, Hemphill, Hill, Howe, Jenkins, Kobayashi, Koudela, Marsh, Mattsson, McAllister, Modry, Omata, Sibley, Speer, Trees, Uggla, Upton, Williams and Lindsay2002; Heydorn and Mehlhorn, Reference Heydorn and Mehlhorn2002).
Comparison of large subunit rDNA and internal transcribed spacer-1 rDNA (ITS-1) sequences derived from H. heydorni, H. hammondi, T. gondii and N. caninum revealed that the genus Hammondia is paraphyletic (Ellis et al. Reference Ellis, Morrison, Liddell, Jenkins, Mohammed, Ryce and Dubey1999). Characterization of alpha-tubulin gene sequences from T. gondii, N. caninum and H. heydorni shows these genes to posses a similar level of DNA sequence similarity amongst each other (Siverajah et al. Reference Siverajah, Ryce, Morrison and Ellis2003). In addition, comparison among introns of the alpha-tubulin gene of different isolates of Hammondia oocysts from canids showed them to be genetically different (Abel et al. Reference Abel, Schares, Orzeszko, Gasser and Ellis2006). Based on the sequence data from ITS-1 and 2 H. heydorni-specific loci, Sreekumar et al. (Reference Sreekumar, Hill, Miska, Rosenthal, Vianna, Venturini, Basso, Gennari, Lindsay and Dubey2004) identified 2 distinct groups among the H. heydorni isolates. In spite of the fact that the nucleotide diversity was low, in both cases the authors suggested that probably more than 1 species exist within the taxon known as H. heydorni.
The Hsp70 comprise a set of chaperones that assist a large variety of protein folding processes in almost all cellular compartments. They all consist of the same working parts: a highly conserved NH2-terminal ATPase domain of 44 kDa and a COOH-terminal region of 25 kDa, divided into a conserved substrate binding domain of 15 kDa and a less-conserved immediate COOH-terminal domain of 10 kDa (Bukau and Horwich, Reference Bukau and Horwich1998). The Hsp70 proteins are essential and highly conserved across prokaryotes and eukaryotes. In fact they are one of the most conserved proteins known to date that is found in all biota (Gupta and Golding, Reference Gupta and Golding1993).
Although Hsp70 is a powerful marker for evolutionary studies of a number of organisms including eukaryotes and prokaryotes (Gupta, Reference Gupta1998; Budin and Philippe, Reference Budin and Philippe1998; Karlin and Brocchieri, Reference Karlin and Brocchieri1998; Sulaiman et al. Reference Sulaiman, Morgan, Thompson, Lal and Xiao2000; Fast et al. Reference Fast, Xue, Bingham and Keeling2002; Slapeta and Keithly, Reference Šlapeta and Keithly2004), evolutionary studies in Apicomplexa using this marker have only been done with Cryptosporidium. The sequence data for the Hsp70 coding genes have provided a useful locus for investigating phylogenetic relationships within this genus (Sulaiman et al. Reference Sulaiman, Morgan, Thompson, Lal and Xiao2000; Zhu et al. Reference Zhu, Keithly and Philippe2000; Xiao et al. Reference Xiao, Sulaiman, Ryan, Zhou, Atwill, Tischler, Zhang, Fayer and Lal2002).
Morrison et al. (Reference Morrison, Bornstein, Thebo, Wernery, Kinne and Mattsson2004) in a comprehensive and consistent phylogeny of the coccidia using the 18S ribosomal coding gene have suggested the analysis using protein coding sequences for phylogenetic purposes in order to produce a species phylogeny of this group of organisms. Their review stimulated the present study of Hsp70 coding genes for evolutionary studies within Hammondia-like organisms, highlighting the genetic diversity among H. heydorni isolates.
MATERIALS AND METHODS
Parasites
Oocysts of T. gondii and H. hammondi were obtained from feces of naturally infected cats, whereas oocysts of N. caninum and H. heydorni were obtained from feces of naturally infected dogs. Canine and feline feces were examined for oocysts by a conventional flotation method, using sucrose solution. Floated material was transferred to a slide and examined by light microscopy. When 10–12 μm sized oocysts were observed, the slide was washed with 1 ml of TE buffer (10 mm Tris-HCl, pH 8·0; 1 mm EDTA, pH 8·0) in sterile Petri dishes. The oocysts were then transferred to 1·5 ml microtubes and washed twice with TE buffer. After the last wash, the supernatant was discarded and the pellet was resuspended in 500 μl of lysis buffer (10 mm Tris-HCl, pH 8·0; 25 mm EDTA, pH 8·0; 100 mm NaCl, 1% SDS). The oocyst suspension was submitted to 3 freeze-thaw cycles and then proteinase K was added at a concentration of 10 μg/ml. The suspension was incubated at 37°C. After overnight incubation the DNA was extracted using a mixture of phenol-chlorophorm, isoamyl-alcohol (25:24:1) and ethanol precipitated as described elsewhere (Sambrook et al. Reference Sambrook, Fritsch and Maniatis1989). DNA of N. hughesi was obtained from tachyzoites of the Oregon strain (Dubey et al. Reference Dubey, Liddell, Mattson, Speert, Howe and Jenkins2001) grown on Vero cells and was kindly supplied by Dr L. F. P. Gondim, Federal University of Bahia, Brazil.
PCR
The nucleotide sequence of the Hsp70 coding gene of T. gondii (U85648) was retrieved from GenBank and submitted to the BLAST search (blastn) in the N. caninum database available in the website of The Institute of Genomic Research (www.tigr.org). A single tentative consensus (TC) sequence of N. caninum was found. Consensus sequences of conserved segments identified within the Hsp70 coding sequence of T. gondii and the TC sequence of N. caninum were used to design 2 primers flanking 1103 bp (HSPF: 5′CAG TCG GAC ATG AAG CAT TGG C3′ and HSPR: 5′ATC GCA CGC TCA CCT TCG TAC AC3′ as sense and reverse primers, respectively). The Hsp70 coding sequence of T. gondii is 2001 nucleotides long (stop codon not included). The primer HSPF anneals from the position 250 to 271, whereas the primer HSPR hybridize from the nucleotide 1330 to 1352. The PCR-Hsp70 cycling conditions used were 94°C for 3 min, followed by 40 cycles of 94°C for 50 sec, 56°C for 50 sec and 72°C for 50 sec. The PCR was finished with a final extension of 72°C for 5 min. The primers, dNTPs and MgCl2 were used at a final concentration of 0·5 μm, 200 μm and 1·5 mm, respectively. Taq DNA polymerase platinum (Invitrogen, Carlsbad, CA) was used at a final concentration of 1·25 Units/50 μl. Five microlitres of the buffer supplied with the enzyme and 5 μl of template DNA were added to the PCR mixture. A second PCR (PCR-ITS1) was carried out employing primers directed to the common toxoplasmatiid 18S and 5·8S rRNA genes. The amplification was performed in 50 μl of final solution as previously described (Rodrigues et al. Reference Rodrigues, Gennari, Aguiar, Sreekumar, Hill, Miska, Vianna and Dubey2004) but using primers JS4 (Slapeta et al. Reference Šlapeta, Koudela, Votýpka, Modrý, Horejs and Lukes2002a) and CT2b (5′TTG CGC GAG CCA AGA CAT C3′, this study). The primers JS4 and CT2b flank about 500 bp within 18S, ITS-1 and 5·8S sequences of the members of the subfamily toxoplasmatinae. The PCR-ITS1 products were directly sequenced and the sequences were submitted to the BLAST search (blastn, www.ncbi.nlm.nig.gov/BLAST) in order to identify the species of each parasite.
Sequencing and sequence alignment
After elution from agarose gel by using a clean-up system (GFX, GE Healthcare, Buckinghamshire, UK) the amplicons of PCR-Hsp70 were sequenced in both directions using the ABI chemistry (ABI PRISM, Foster City, CA) with the sense and reverse primers. A third sequencing primer (HSPFII: 5′GAA CGT CCT CAT CTT CGA CAT GG3′, from the position 582 to 604 within the Hsp70 encoding gene of T. gondii) internal to the fragment of 1103 bp was designed in order to sequence the internal segment of the amplicon. Each strand was sequenced at least 4 times in order to increase the reliability of the results. The sequences were assembled and the contig formed with the phred-base calling and the phrap-assembly tool available in the suite Codoncode Aligner v.1.5.2 (CodonCode Corp. Dedham, MA, USA). The assemblies were recovered with each residue score equal or greater than 20. The amplicons of PCR-ITS1 were sequenced exactly as above but using the oligonucleotides JS4 and CT2b as sequencing primers. The nucleotide sequences were aligned by using the software ClustalX v.1.83 (Thompson et al. Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997).
Phylogenetic analysis
The aligned sequences of each allele of Hammondia-like organisms were used as input to the program PAUP* v.4.0b10 (Sinauer Associates, Inc.). Phylogenies were reconstructed with maximum likelihood (ML) and maximum parsimony (MP). For the ML analyses the evolutionary model of nucleotide substitution was calculated by using the software Modeltest v.3.7 (Posada and Crandall, Reference Posada and Crandall1998) using the hierarchical likelihood ratio tests (hLRTs). The trees of MP analysis were searched with the branch and bound search and the characters were considered unordered. The trees reconstructed with MP were bootstrapped with heuristic search and 1000 replicates whereas the data obtained with ML were bootstrapped with 100 replicates. The final trees were drawn using TreeView version 1.6.6 (Page, Reference Page1996). In the Hsp70 phylogeny, sequences of Cyclospora and Eimeria were used to serve as outgroup.
Nucleotide diversity within Apicomplexa
Hsp70 coding sequences of one representative of each species of Hammondia-like organisms and several other homologue sequences available in the GenBank databases were used in this study (Table 1). The sequences from GenBank were obtained by using the translating BLAST tool (tblastx, www.ncbi.nlm.nig.gov/BLAST) and the coding sequence of Hsp70 of T. gondii (U85648) as query. Nucleotide sequences from the following members of Apicomplexa were retrieved: Babesia, Cyclospora, Cryptosporidium, Plasmodium and Theileria. This search did not find any sequence from Sarcocystis. The nucleotide diversity within different groups of sequences was computed with DnaSP v.4.10 (Rozas et al. Reference Rozas, Sanchez-Del-Barrio, Messeguer and Rozas2003).
Taxons used in the nucleotide diversity analysis and average number of nucleotide differences per site between two sequences (Pi) in each group of partial Hsp70 coding sequences
1 Accession number in GenBank (www.ncbi.nlm.nih.gov).
2 s.d., Standard deviation of Pi.
RESULTS
We sequenced 951 nucleotides of the Hsp70 genes (corresponding to 317 aminoacids) and the complete ITS-1 rDNA sequences from 14 H. heydorni isolates, 2 H. hammondi isolates, 3 N. caninum isolates, 6 T. gondii isolates and 1 N. hughesi isolate. In spite of the fact that we used uncloned field isolates, no DNA polymorphism was observed in any of the chromatogram files.
Multiple sequence alignments of the Hsp70 genes revealed 6 alleles of H. heydorni, 1 allele of H. hammondi, 1 allele of N. caninum, 1 allele of N. hughesi and 2 alleles of T. gondii (Table 2). No gap was encountered. The 2 alleles of T. gondii differ by 2 synonymous substitutions. Only 2 synonymous substitutions are responsible for the difference between the alleles of N. hughesi and N. caninum.
Hsp70 and ITS-1 alleles from Hammondia heydorni, Toxoplasma gondii, Neospora caninum, Neospora hughesi and Hammondia hammondi
1 Isolates of Hammondia heydorni (1–14); isolates of Toxoplasma gondii (15–20); isolates of Neospora caninum (21–23); isolate of Neospora hughesi (24); isolates of Hammondia hammondi (25–26).
2 Alleles of Hsp70 (each different allele is numbered with roman characters).
3 Alleles of ITS-1 (each different allele is numbered with roman characters).
4 GenBank Accession numbers of Hsp70 coding sequences.
5 GenBank Accession numbers of ITS-1 sequences.
6 Genetic groups of H. heydorni isolates, ND: genetic groups not determined for other species than H. heydorni.
The alignment of the ITS-1 rDNA sequences revealed 5 alleles of H. heydorni, 1 allele of H. hammondi, 2 alleles of N. caninum, 1 allele of N. hughesi and 2 alleles of T. gondii (Table 2). The 2 ITS-1 alleles of N. caninum differ by 1 replacement. Only 1 transition contributes to the difference between the 2 alleles of T. gondii. The ITS-1 alleles of N. caninum and N. hughesi differ by 7 or 8 nucleotides and 1 dinucleotide indel.
The number of nucleotide differences between each pair of alleles from H. heydorni sequences is shown in Tables 3 and 4. The differences between the alleles of H. hammondi and the homologous sequences of T. gondii strain RH (U85648 and X75429) are 24 and 14 residues at the Hsp70 and ITS-1 loci, respectively.
Number of nucleotide differences between each pair of ITS-1 alleles from Hammondia heydorni
Number of nucleotide differences between each pair of Hsp70 alleles from heydorni
The Hsp70 predicted amino acid sequences of T. gondii and H. hammondi are identical. The Hsp70 predicted sequences of N. caninum, N. hughesi and H. heydorni have 100% of identity. The difference between Hsp70 sequences of N. caninum/N. hughesi/H. heydorni and T. gondii/H. hammondi is only 1 conservative substitution (Asp→Glu).
For the ML analyses, the selected model of nucleotide substitution assumed equal base frequencies, equal transversion frequencies, variable transition frequencies and Gamma distributed rate variation among sites (this model is named TrNef+G) (Tamura and Nei, Reference Tamura and Nei1993). In Fig. 1 the phylogenetic trees show 2 well-supported clades grouping the H. heydorni. One of these clades is formed by the Hsp70 alleles I and II. The other clade is compound by the alleles IV, V and VI. The position of allele III is uncertain.
Phylogenetic relationships among the Hammondia-like organisms inferred from the alignment of the Hsp70 coding employing Maximum Likelihood (ML) and Maximum Parsimony (MP) analysis (panels A and B, respectively). The trees are rooted with Cyclospora colobi (Cco, DQ062460.1), C. cayetanensis (Cca, AY240875.3) and Eimeria acervulina (Eac, Z26134.1). The numbers indicate the number of times that the branches are supported after 100 (ML analyses) and 1000 (MP analyses) bootstrap replicates. The branch lengths in the ML tree are proportional to the amount of evolutionary changes. Only 1 MP tree with 607 steps was found.
The branching order within the Hammondia-like organisms is not well resolved. Neospora caninum, N. hughesi, H. hammondi and T. gondii branch together to the exclusion of Hammondia heydorni in the ML and MP analyses but with low statistic support (Fig. 1, panels A and B).
The 6 Hsp70 alleles and 5 ITS-1 alleles revealed the existence of 8 haplotypes among the isolates of H. heydorni which were separated into 3 genetic groups, named A, B and C (Table 2).
Multiple sequence alignments of the Hsp70 genes of the organisms presented in Table 1 did not reveal any gap within the sequences. The average number of nucleotide differences per site between 2 sequences (Pi) in each group of sequences was calculated and the values can be found in Table 1. Although the Hammondia-like group is predominantly compound by distinct genera, this group presented the lowest value of nucleotide diversity (0·092). The highest nucleotide diversity was encountered in the group formed by the Theilerias (0·197).
DISCUSSION
Although widely used for inferring phylogenies of a number of organisms, the Hsp70 coding sequences should be used with caution for the evolutionary studies of Apicomplexa. The first codons of the 5′terminal region and the 3′terminal region encoding a 10 kDa putative substrate-stabilization domain (Bukau and Horwich, Reference Bukau and Horwich1998) produced poor alignments as many gaps have to be assumed. The 3′terminal region of Hsp70 coding sequences of some parasites is known to code for repeat units that vary in the number of copies (Sheppard et al. Reference Sheppard, Kemp, Anders and Lew1989; Sharma, Reference Sharma1992; Lyons and Johnson, Reference Lyons and Johnson1998). The number of such repeats varies even among sequences from the same species (Lyons and Johnson, Reference Lyons and Johnson1998). To overcome this problem we analysed a fragment that starts after and ends before those regions and spans 951 nucleotides within part of the conserved ATPase and substrate-binding site domains.
Considering the infection by coccidians, there is the possibility that the oocysts were from animals infected with multiple species or multiple lineages of a single species. However, this possibility was ruled out because no DNA polymorphisms were observed in any of the sequence files, suggesting that there were no indications for mixed infections.
Considering the Hsp70 gene phylogeny of the Hammondia-like organisms, at least 2 distinct genetic lineages could be recognized within the clade formed by the H. heydorni species. A third lineage may exist if one considers the differences between the allele III and the alleles IV, V and VI. In the ML tree, the allele III is grouped together with alleles IV, V and VI while MP tree separated the allele III from the other alleles. In both cases, the position of allele III has low statistic support. The 6 Hsp70 alleles and 5 ITS-1 alleles revealed the existence of 8 haplotypes among the isolates of H. heydorni. In fact, a notable similarity of the haplotypes at each of the 2 loci was observed and the isolates could be separated into 3 genetic groups named A, B and C. ‘Recombinants’ are found only within each distinct genetic group. Obviously, these conjectures are valid if one considers that the population structure of H. heydorni is clonal. The clonality of such a population remains to be elucidated.
Based on 2 H. heydorni specific loci (HhAP07 and HhAP10) and ITS-1 rDNA sequences, Sreekumar et al. (Reference Sreekumar, Hill, Miska, Rosenthal, Vianna, Venturini, Basso, Gennari, Lindsay and Dubey2004) also found at least 2 distinct genotypes among their isolates of H. heydorni. Some of the isolates analysed by these authors were also evaluated here and in both cases such isolates were clustered identically. The isolate HheV5, named Brazil-1 by Sreekumar et al. (Reference Sreekumar, Hill, Miska, Rosenthal, Vianna, Venturini, Basso, Gennari, Lindsay and Dubey2004), was the only taxon classified into genetic group C because it proved to be quite divergent from those of groups A and B at Hsp70 loci.
The nucleotide diversity existing between the 2 species of Neospora at Hsp70 loci is lower than that observed at ITS-1. The differences between the 2 lineages of H. heydorni at ITS-1 loci level are comparable to that existing between N. caninum and N. hughesi. But at Hsp70 loci, the nucleotide diversity among H. heydorni lineages is much higher than that existing between the 2 species of Neospora. Thus, if N. caninum and N. hughesi are considered different species, the 2 genetically distinct lineages of H. heydorni would be separate species of a single genus, based on genetic criteria.
The phylogenetic analyses presented here indicated that T. gondii and H. hammondi are sister taxons, rendering the genus Hammondia paraphyletic, thus confirming previous observations (Ellis et al. Reference Ellis, Morrison, Liddell, Jenkins, Mohammed, Ryce and Dubey1999). The comparison of Hsp70 partial coding sequences from H. heydorni, T. gondii and N. caninum revealed these genes to possess a similar level of evolutionary distance. In fact, the placement of Neospora spp. as sister group of T. gondii/H. hammondi remains difficult to demonstrate as the Hsp70 gene trees did not resolve the relationships between the major Hammondia-like groups with confidence. Ellis et al. (Reference Ellis, Morrison, Liddell, Jenkins, Mohammed, Ryce and Dubey1999) and Mugridge et al. (Reference Mugridge, Morrison, Jakel, Heckeroth, Tenter and Johnson2000) faced the same problem when they reconstructed the Toxoplasmatinae phylogeny using the large subunit rDNA sequences. This lack of resolution may be explained by the hypothesis that these groups emerged as a result of radiation, which may be explained as a burst process of multiple simultaneous speciation events (Hoelzer and Melnick, Reference Hoelzer and Melnick1994). However, the robustness of such a hypothesis would increase when more genes are analysed.
The study of the nucleotide diversity of Hsp70 coding sequences within some groups of Apicomplexa revealed the Hammondia-like group as one of the less divergent groups. The low variability existing among Hammondia-like organisms has been already described with 18S and ITS-1 rDNA genetic sequences (Ellis et al. Reference Ellis, Morrison, Liddell, Jenkins, Mohammed, Ryce and Dubey1999; Jenkins et al. Reference Jenkins, Ellis, Liddell, Ryce, Munday, Morrison and Dubey1999; Mugridge et al. Reference Mugridge, Morrison, Heckeroth, Johnson and Tenter1999, Slapeta et al. Reference Šlapeta, Modrý, Kyselová, Horejs, Lukes and Koudela2002b; Sreekumar et al. Reference Sreekumar, Hill, Miska, Rosenthal, Vianna, Venturini, Basso, Gennari, Lindsay and Dubey2004; Morrison et al. Reference Morrison, Bornstein, Thebo, Wernery, Kinne and Mattsson2004).
Based on phylogenetic analysis, 3 plausible solutions for the nomenclature of the Hammondia-like organisms were outlined by Slapeta et al. (Reference Šlapeta, Modrý, Kyselová, Horejs, Lukes and Koudela2002b). While it is now evident that the majority of researchers agree that the Hammondia-like organisms are formed by at least 5 species, the question that remains is how many genera need to be kept. The mounting evidence from nucleotide diversity and phylogenetic analysis of Hsp70 locus supports the first scenario, which would retain only a single genus. The phylogenetic analysis of Hsp70 coding genes also supports the view that 3 distinct clades would be classified as genera Toxoplasma (T. gondii and H. hammondi), Neospora (N. caninum and N. hughesi), and a new genus to accommodate the 2 variants of H. heydorni (the second scenario). According to our data, the third scenario outlined by Slapeta et al. (Reference Šlapeta, Modrý, Kyselová, Horejs, Lukes and Koudela2002b) i.e. grouping the Hammondia-like organisms based on the host (feline isolates are Toxoplasma and canine isolates are Neospora) should be discarded.
Similar to rRNA, the Hsp70 is present in all species and performs an essential function within the cell. Thus, comparative genealogies using both markers are desirable in order to infer the true species phylogenies and produce a robust taxonomy, not only for Hammondia-like organisms but probably for any taxonomic level.
The choice of Hsp70 coding sequences for phylogenetic reconstruction of this group of organisms presented some advantages. Firstly, it allows the inclusion of outgroups with high confidence and lower risk to the long-branch attraction artifact due to the low distance of the ingroup taxa to outgroup. An important criterion for the reliability of a marker is its sensitivity to the long-branch attraction artifact which is decreased when the distance to the outgroup decreases (Felsenstein, Reference Felsenstein1978) and the use of an appropriate outgroup is needed because this determines the root of the trees. We used the Hsp70 genetic sequence from Cyclospora spp. and E. acervulina as outgroup because they are the most closely related of the Hammondia-like organisms with Hsp70 coding sequences available in GenBank.
Secondly, because of the similar size of this gene in various species and its high degree of conservation, a reliable alignment from several species was readily obtained. It is noteworthy to stress that no gaps where observed in the alignment produced with the Hsp70 homologous fragments of the apicomplexans, hence phylogenies using such data require minimal or no a priori assumptions. As stated before, the alignment of homologous positions in a set of sequences is the starting point in phylogenetic analyses from which all inferences are derived (Gupta, Reference Gupta1998). Finally, the alignment of Hsp70 sequences from closely related species shows many conserved regions that may serve as anchorage site for consensus primers, which can be successfully used for PCR amplification of a wide range of organisms. With aligned sequences of N. caninum and T. gondii we were able to design consensus primers capable of amplifying Hsp70 genetic sequences of H. hammondi, H. heydorni and N. hughesi.
The authors extend thanks to FAPESP for financial support, grant numbers 04/04040-8 and 04/04041-4.
