1 Department of Veterinary Preclinical Science, University of Liverpool, Brownlow Hill and Crown Street, Liverpool L69 7ZJ, UK
2 Department of Veterinary Pathology, University of Liverpool, Leahurst, Neston, South Wirral CH64 7TE, UK
3 Department of Veterinary Clinical Science, University of Liverpool, Leahurst, Neston, South Wirral CH64 7TE, UK
Correspondence
N. H. Ogden
ogdenn{at}courrier.umontreal.ca
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ABSTRACT |
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The GenBank accession numbers for the sequences determined in this work are given in the text.
Present address: Groupe de Récherche en Épidémiologie et Santé Publique, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, CP 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6.
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INTRODUCTION |
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A. phagocytophilum [formerly named as the agent of human granulocytic ehrlichiosis (aoHGE), Ehrlichia phagocytophila and Ehrlichia equi: Ristic & Huxsoll, 1984; Dumler et al., 2001
] can infect a very wide range of species (wild rodents and deer, cats, dogs, llamas and humans: Ogden et al., 1998
) and some isolates can cross-infect between species (Foley et al., 2002
). Some isolates from one species do not seem to be directly infective for another (Pusterla et al., 2001
), however, and early cross-protection studies using European isolates from sheep (i.e. previously E. phagocytophila), suggested that a high degree of strain diversity may occur amongst different isolates of A. phagocytophilum (Foggie, 1951
; Tuomi, 1967
).
Such conflicting observations could in part be explained by the characteristics of the immunodominant 44 kDa protein (P44) of A. phagocytophilum, which is encoded by a multigene family (p44; Murphy et al., 1998). Expression of different paralogues occurs in different environments such as mammalian host and vector tick cells (Zhi et al., 2002a
, b
), and differential expression is also thought to be responsible for antigenic variation in the host (Barbet et al., 2003
). The discovery of this property of the bacterium raises the hypothesis that evidence for strain diversity amongst A. phagocytophilum isolates observed in some experiments could have been due to phenotypic rather than genetic variations. Nevertheless, the close interaction of expressed p44 proteins with host cells (Park et al., 2003
) may mean that some co-evolution of this gene with different reservoir hosts in different foci (e.g. rodents in the USA as opposed to sheep in the UK) has occurred. This could have consequences for the potential infectivity of A. phagocytophilum, in infected ticks in different foci of infection, for humans or domesticated animals.
Some studies have already suggested that variations in either 16S rRNA gene or p44 sequences from USA isolates may be associated with ecological or clinical characteristics of A. phagocytophilum (Carter et al., 2001; Massung et al., 2002
). In the present study, we have investigated the hypothesis that differences in either 16S rRNA gene or p44 sequences may occur amongst UK isolates from ruminants (that have yet to be associated with human disease), and isolates from other parts of the world, particularly the USA, where human disease does occur.
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METHODS |
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Amplification and analysis of 16S rRNA gene sequences.
DNA was extracted from 200 µl of each blood sample used in the study using DNeasy blood and tissue kits (Qiagen). DNA was extracted from the engorged tick by alkaline digestion as previously described (Ogden et al., 2002). A nested PCR was used to amplify a 546 bp region of the 16S rRNA gene of A. phagocytophilum (Massung et al., 1998
). Negative controls were incorporated at a rate of one per test sample. Purified PCR products (Wizard PCR Preps DNA Purification system; Promega) were directly sequenced with primers ge9f and ge2 (Massung et al., 1998
) according to the manufacturer's instructions (ABI prism dye terminator cycle sequencing ready reaction kit; Perkin Elmer). 16S rRNA gene sequences from this study were compared to published sequences of 19 isolates of A. phagocytophilum from Europe, USA and Asia, and to sequences from related bacteria. All sequences were aligned using the PILEUP program of GCG (Wisconsin Sequence Analysis Package, Genetics Computer Group, version 8.1). Phylogenetic analysis of the aligned sequences was performed using the programs NEIGHBOR, DNADIST (kimura) and SEQBOOT from the PHYLIP package (Felsenstein, 1989
). Trees were drawn using TREEVIEW (Windows version 32; Page, 1996
).
Amplification and analysis of p44 sequences.
Amplicons of p44 gene paralogues of approximately 550 bp were obtained from the DNA samples using primers P3708 and P4257, which anneal to the N- and C-terminal conserved regions of p44 paralogues in USA isolates, flanking the central hypervariable region of the genes, as previously described (Zhi et al., 1999). PCR products were cloned into the pCRII plasmid and transformed into Escherichia coli using a pCR 2.1-TOPO TA cloning kit (Invitrogen). Plasmids containing appropriately sized inserts (400600 bp) were identified using standard protocols, purified (Wizard Plus SV Miniprep DNA Purification System; Promega) and their inserts were sequenced using primers T4 and SP7. The DNA sequences obtained were aligned in GCG with all (65 in number) of the available, published p44 paralogue gene sequences of A. phagocytophilum (Caspersen et al., 2002
; Lin et al., 2002
; Zhi et al., 1999
, 2002b
), including those of the Webster and BDS strains, and the NY31, NY36, NY37, HZ and LL isolates from the USA (GenBank accession nos AF443396 to AF443413 and AF443415 to AF443419, AF059181, AF135263, AF135254 to AF135257, AF412818 to AF412831, AF414591 and AY064513 to AY064530).
A number of comparisons of relatedness' amongst p44 paralogues from the UK and USA isolates were made. First, variations in similarity of different parts of the sequenced UK paralogues were investigated using the PILEUP and PLOTSIMILARITY programs in GCG, and a consensus sequence of paralogues from UK isolates was generated. Antigenicity and hydrophobicity of different parts of this consensus sequence were investigated using the PLOTSTRUCTURE program in GCG for a qualitative comparison with p44 paralogues from USA isolates (Lin et al., 2002). Second, the percentage similarity of each UK sequence obtained in the present study, to each other and to published p44 sequences from USA isolates, was estimated from the DISTANCES program in GCG. From these, uncorrected distance trees of DNA sequences were developed. Due to a very high number of insertions and deletions, alignments of these sequences for phylogenetic analyses that could yield statistical support for tree topology were impossible (see Results). We did, however, investigate whether any paralogues had particularly high similarity (>90 %) to any others and whether such similar paralogues were from isolates from the same or different countries. Third, we investigated the sites of base substitutions amongst paralogues that were more than 90 % similar to another, using the web-based SNAP software (http://www.hiv.lanl.gov; Korber, 2000
). The numbers of non-synonymous substitutions per base in the N- and C-terminal conserved regions of these highly similar paralogue pairs or groups were estimated and then compared with the numbers of non-synonymous substitutions per base in the central hypervariable regions using the non-parametric MannWhitney U test.
GenBank accession numbers
Partial 16S rRNA sequences.
North Wales sheep isolate AY149635, North Wales tick isolate AY149637, AB strain AY176586, Cairn strain AY176587, Feral Goat strain AY176588, Harris strain AY176589, Lephimore strain AY176590 and OS strain AY176591.
Partial p44 sequences.
AB strain paralogue AB1 AY176512. Cairn strain paralogues Cairn1, Cairn2, Cairn3, Cairn4, Cairn6, Cairn7, Cairn8, Cairn9, Cairn10, Cairn11, Cairn12, Cairn13, Cairn15, Cairn16 and CairnB01 AY176513 to AY176528 respectively. Bovine isolate paralogues Cow1, Cow2 and Cow3 AY176529 to AY176531 respectively. Feral Goat strain paralogues FGA1, FGA3, FGA4, FGA5, FGA6, FGA7, FGA8, FGA12, FGA13, FGA14, FGA15, FGA17, FGA18, FGA19, FGA20, FGA21, FGA22, FGA23, FGA24, FGA25, FGA26, FGA27, FGA28, FGA29, FGA30, FGA31, FGA32, FGB1, FGB2, FGB4, FGB6, FGB10 and FGB11 AY176532 to AY176564 respectively. Lephimore strain paralogues Lep1, Leph2 and Leph4 AY176565 to AY176567. North Wales sheep isolate paralogues NW2, NW3, NW5, NW7, NW8, NW9, NW11, NW12, NW13, NW14, NW16, NWB1 and NWB2 AY176568 to AY176581 respectively. North Wales tick isolate paralogue NWT1, AY176582.
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RESULTS |
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All in-frame sequences, as well as the consensus, yielded putative protein sequences similar to those of sequences from USA isolates, i.e. two moderately hydrophobic N- and C-terminal conserved regions of relatively low potential antigenicity flanking a more hypervariable, potentially hydrophilic and antigenic region that comprised three domains separated by highly conserved amino acids. The highly conserved amino acids that delineated the hypervariable region domains were the same as those identified in USA isolates (Lin et al., 2002). In general, N- and C-terminal conserved regions were those most highly conserved amongst paralogues from all UK and USA isolates. These results are summarized in Fig. 2
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DISCUSSION |
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Sequences of p44 genes, which were similar to those from USA isolates, were amplified from all of the tested UK strains' and isolates. Up to 20 paralogues of the p44 gene have been discovered in the USA isolates that have been examined in detail to date (Caspersen et al., 2002). We did not exhaustively sequence the UK isolates but in one isolate, only six sequenced clones were identical amongst 37, indicating that it most likely had a higher number of p44 paralogues. Some UK A. phagocytophilum isolates could, therefore, have a larger complement of different paralogues than their USA counterparts. This phenomenon may have been due to the occurrence of mixed infections in sheep with different genotypes' of A. phagocytophilum as detected in other studies by 16S rRNA gene sequence analysis (Stuen et al., 2002
). Mixed infections could have occurred, however, in the USA isolates studied to date. Similarities amongst and between paralogues from USA and UK isolates were mostly maintained at the putative peptide level and only a very low number of paralogues were out of frame, suggesting that functional integrity of a large number of possible p44 proteins is important for bacterial survival.
Most p44 sequences had only moderate similarity to one another (75 % or less) and there was no clear clustering of variants from individual UK and USA isolates, or between isolates, in uncorrected distance trees. Some sequences did, however, form groups with greater than 90 % similarity (the similarity groups), comprising in some cases paralogues from the same isolate, in some cases different isolates from the same countries and host species, and in one case, isolates from different countries and host species. Much of the variation amongst paralogue sequences was due to insertions and deletions of variable size (mostly in the hypervariable domains), which made impossible the meaningful alignments required for statistically supported phylogenetic analyses (Felsenstein, 1989). We cannot, therefore, formally assign confidence limits to the distance tree topology. It should be noted though, that except for two sequences with between 80 and 90 % similarity to members of a similarity group, nearest neighbours to similarity groups were all less than 80 % similar to any members of that group. However many times the tree is redrawn, therefore, members of the same similarity group will always cluster together. Furthermore, were the similarity groups to have occurred by random chance (in the occurrence of insertions, deletions and substitutions), variation amongst paralogues of the same similarity group should have either occurred randomly along the length of the gene, or at least followed the general pattern of variation amongst p44 paralogues in having more variable central regions with more conserved flanking regions. The precise opposite was observed: paralogues within similarity groups' had significantly higher similarity in their central domains compared to that occurring in the flanking regions. Paralogues of similarity groups were, therefore, different to other paralogues in both the degree of similarity and the pattern of variation in the different gene regions. The central hydrophilic domains are those most likely to form ligands with host cells (Lin et al., 2002
; Zhi et al., 2002a
), and high similarity in these regions suggests functional similarity amongst the members of each similarity group. This is more in line with the observation that at least some p44 paralogues are involved in specific interactions with host or vector cells that are important for survival of A. phagocytophilum (Park et al., 2003
). Such paralogues would be more likely to be conserved if the bacterium were to remain infective for the same host species.
The predominantly high degree of variation we observed amongst p44 sequences from UK sheep-derived isolates underlines the potential for phenotypic variation of A. phagocytophilum even within foci of infection. The existence of similarity groups, however, suggests that a few paralogues are more highly conserved amongst isolates, which, being potentially important for adherence to host cells, may provide insight into the host or vector species that an isolate is capable of infecting. In the sample of sequences available to us, similarity groups' were nearly always country specific (even though paralogues from UK isolates were amplified using primers known to amplify paralogues in USA isolates) and more likely to occur amongst USA rather than UK isolates. At most, these findings may suggest that indeed some specific p44 paralogues are conserved and adapted to different reservoir hosts or vectors in different geographical regions, with corresponding differences in infectivity for different non-reservoir vertebrate species such as humans. Further studies are required, however, to increase the sample size of paralogues and hosts of origin from both Europe and North America to confirm this. At least, our findings suggest that the USA isolates investigated to date (mostly from humans) contained a lower diversity of p44 paralogue variants than did UK isolates from sheep, i.e. the potential for phenotypic variation of A. phagocytophilum isolates infecting humans may be considerably less than that occurring in nature. We did find one similarity group that comprised paralogues from ruminant UK and human USA isolates, which was the p44-18 group. It has been hypothesized that the p44-18 paralogue may have a somewhat specific role in the initial phase of infection of previously naïve animals and humans (Zhi et al., 2002b).
In summary, partial 16S rRNA gene sequences did not, for the most part, discriminate amongst UK sheep isolates and isolates from other species (including humans suffering clinical HGE) in other countries. Partial p44 paralogues were amplified from all UK isolates and these had similarities in their DNA and putative peptide sequences, with p44 paralogues amplified from isolates from humans and horses in the USA. A smaller number of paralogues formed similarity groups, which, in the sample available to us, were more common amongst USA isolates and rarely comprised paralogues from isolates from both the UK and the USA. As paralogues of similarity groups were particularly similar in gene regions likely to interact with the host, they may be useful targets for investigations and identification of any adaptations of A. phagocytophilum isolates to different vertebrate host species. Further study of similarity groups of p44 paralogues may, therefore, be fruitful in predicting the infectivity for humans, of different field isolates of A. phagocytophilum.
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ACKNOWLEDGEMENTS |
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Received 15 July 2003;
revised 23 October 2003;
accepted 8 December 2003.
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