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First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria
Research in Veterinary Science 94 (2013) 27–32
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First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria Joshua Kamani a, Chung-Chan Lee b, Ayuba M. Haruna c, Ping-Jun Chung b, Paul R. Weka a, Yang-Tsung Chung b,⇑ a
Parasitology Division, National Veterinary Research Institute PMB 01, Vom, Plateau State, Nigeria Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan c State Veterinary Hospital Jos, Ministry of Agriculture and Natural Resources, Plateau State, Nigeria b
a r t i c l e
i n f o
Article history: Received 28 March 2012 Accepted 28 July 2012
Keywords: Ehrlichia canis Dogs PCR Phylogenetic analysis TRP36 gene Nigeria
a b s t r a c t The present study aimed to detect the presence of Ehrlichia canis in naturally infected dogs in Nigeria, using a combination of PCR and sequence analysis of the 16S rRNA gene and two genes encoding the tandem repeat-containing proteins (TRPs), TRP19 and TRP36. Out of a total of 100 blood samples collected from domestic dogs presented to veterinary hospitals in Jos, the capital city of Plateau State of Nigeria, 11 were positive in nested PCR for E. canis. Sequencing results for these amplicons showed that all of the 16S rDNA sequences (1623 bp) or the TRP19 coding sequences (414 bp) were identical to each other and had very high similarities (99.3–100%) with those from other E. canis strains accessible in GenBank. The TRP36 gene sequences derived from the 11 Nigerian isolates were identical to each other except for the number of the 27-bp repeat unit in a tandem repeat region, which was found to be 8, 12 or 18. Without considering the number of tandem repeats, these sequences had 100% identity to that of the reported Cameroon 71 isolate, but distinctly differed from those obtained from other geographically distant E. canis strains previously published. A phylogenetic tree of E. canis based on the TRP36 amino acid sequences showed that the Nigerian isolates and the Cameroon 71 isolate fell into a separate clade, indicating that they may share a common ancestor. Overall, this study not only provides the first molecular evidence of E. canis infections in dogs from Nigeria but also highlights the value of the TRP36 gene as a tool to classify E. canis isolates and to elucidate their phylogeographic relationships. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Canine monocytic ehrlichiosis (CME) caused by Ehrlichia canis is of veterinary importance worldwide. The causative agent of the disease is a Gram negative, obligatory intracellular bacterium, which is enveloped with a rippled thin outer membrane (Rikihisa et al., 1997; Boozer and Macintire, 2003). E. canis has special tropism for monocytes and macrophages (Huxsoll et al., 1970). It consists of a single circular chromosome containing 1,315,030 nucleotides (Mavromatis et al., 2006). The agent is predominantly transmitted by the brown dog tick Rhipicephalus sanguineus (Dantas-Torres, 2008; Stich et al., 2008). CME has been reported throughout the world, with a higher frequency in tropical and subtropical regions (Vinasco et al., 2007; Rar and Golovljova, 2011). Diagnosis of the disease can be challenging due to its different phases and multiple clinical manifestations (Harrus and Waner, 2011). Traditional diagnostic techniques such ⇑ Corresponding author. Address: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. Tel.: +886 4 2284 0751; fax: +886 4 2286 2073. E-mail address: [email protected] (Y.-T. Chung). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.07.031
as hematology, cytology, serology and isolation are valuable diagnostic tools for CME. However, such methods are labor-intensive and the results obtained are often ambiguous because of their low sensitivities or inability to differentiate past exposure to the disease or active infections (Breitschwerdt et al., 1998). The definitive diagnosis of E. canis infection therefore requires molecular techniques (Harrus et al., 1998; Harrus and Waner, 2011) especially the polymerase chain reaction (PCR), which has been used extensively for specific diagnosis of tick-borne protozoal and ehrlichial infections in dogs in developed countries (Gal et al., 2008). This is not the case with most developing countries due to decay in infrastructure and lack of expertise to conduct such tests. In Nigeria, the diagnosis of infections with Ehrlichia species is usually based on the detection of pathogens in peripheral blood under a microscope. The search for morulae by microscopy is difficult, time-consuming and has been estimated to be successful in only about 4% of cases (Woody and Hoskins, 1991). Such challenges in the diagnosis of ehrlichiosis in dogs in Nigeria have led to wrong conclusion among veterinarians that the disease rarely occur, and thus, is not considered a major problem. As such there has not been any published report on molecular status of CME agent in Nigeria in the last two decades with no information
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available on its DNA sequence and phylogeny. The purpose of this study was thus to assess the presence of E. canis in dogs in Nigeria using molecular methods. We sought to amplify with PCR the 16S ribosomal RNA gene (16S rDNA) and two genes encoding the tandem repeat-containing proteins (TRPs), TRP19 (formerly gp19) and TRP36 (gp36), characterized by previous studies (Doyle et al., 2005; Hsieh et al., 2010) followed by sequencing of those fragments. We also conducted sequence alignment and phylogenetic analysis to identify E. canis genotypes. 2. Materials and methods
clones were picked and purified individually using the QIAprep Spin Miniprep Kit (Qiagen), and then sequenced by using an ABI PRISM 3730 capillary sequencer (Applied Biosystems) and the Dye Terminator Cycler Sequencing Kit (Applied Biosystems) with a vector primer (T7 or SP6) and an appropriate internal sequencing primer (see Table 1). Both the sense and antisense strands of each PCR amplicon were sequenced. Sequences were manually edited to resolve ambiguities. A consensus sequence was obtained for each amplicon by comparing both the sense and antisense sequences. Representative nucleotide sequences for the E. canis genes reported here have been deposited in the GenBank database under accession numbers JN622141–3 and JN982336–41.
2.1. Collection of blood samples and DNA extraction 2.3. Sequence and phylogenetic analyses Blood was collected from 100 dogs manifesting clinical signs of tick-borne diseases that were referred to veterinary hospitals in Jos, the capital city of Plateau State of Nigeria, from February to July 2011. Blood was collected from cephalic vein into sterile tubes with anticoagulant (EDTA), and held at 4 °C until arrival at the laboratory for further processing. Genomic DNA was extracted from 200 ll of whole blood samples by using AxyPrep TM Genomic DNA extraction kit (Axygen Biosciences, Axygen Scientific, Inc. USA) according to manufacturer’s recommendation. Nucleic acid was eluted into 100 ll of elution buffer and stored at 20 °C until further use. 2.2. PCR amplification and sequencing Primers used for the amplification and sequencing of the 16S rRNA, TRP19 and TRP36 genes of E. canis (Cardenas et al., 2007; McBride et al., 2007) were designed using primer design software (Primer Select, DNASTAR, Madison, WI, USA) and E. canis genome sequence information (Mavromatis et al., 2006) and are presented in Table 1. PCR amplification was carried out as described previously (Hsieh et al., 2010). The resulting PCR products were electrophoresed on a 1.2% agarose gel stained with ethidium bromide to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 kb Plus DNA Ladder, Promega). In each case, the single amplicon of the expected size was column purified using the QIAquick PCR Purification Kit (Qiagen) and ligated into the pGEM-T vector (Promega) for subsequent transformation in the Escherichia coli DH5a competent cells. For each PCR amplicon, at least three recombinant
The BLAST program (http://www.ncbi.nlm.nih.gov/ BLAST) was used for the comparison and the analysis of sequence data obtained in this study with those previously deposited in GenBank. Multiple sequence alignment was conducted using the program AlignX (Vector NTI Suite V5.5, InforMax, North Bethesda, MD, USA) with an engine based on the Clustal W algorithm (Thompson et al., 1994). A TRP36 phylogenetic tree was inferred using the neighbor-joining method as implemented by MEGA software version 4 (Tamura et al., 2004). The distance matrix of amino acid divergences was calculated according to Kimura’s two parameter model furnished by MEGA. A bootstrap resampling technique of 1000 replications was conducted to statistically support the reliabilities of the nodes on the tree. 3. Results All blood samples were subjected to DNA extraction followed by nested PCR amplification with the 16S rDNA-targeted primers (see Table 1). Among 100 samples examined, 11 (11%) were positive for E. canis. Sequencing results for those amplicons showed that all 16S rDNA sequences of 1623 base pairs (bp) derived from 11 Nigerian isolates were identical to each other and bore 99.4– 100% identities with the corresponding sequences from E. canis strains in other geographic areas (Table 2). This is in agreement with previous investigations (Aguirre et al., 2004; Siarkou et al., 2007; Yu et al., 2007; Pinyoowong et al., 2008), indicating that the genetic profile of canine E. canis strains based on the 16S rDNA sequence was highly conserved.
Table 1 Primers used for the amplification and sequencing of the 16S rRNA, TRP19 and TRP36 genes from the Nigerian E. canis isolates. Target
Primera
Sequence
16S rDNA
Ec16S-F1 Ec16S-R1 Ec16S-F2 Ec16S-R2 Ec16S-F3 Ec16S-R3
50 -TCA ACC CAT GGC TAA ATG TCA-30 50 -CAG ATG TGG AGA TAA AGG CCT-30 50 -GGT AGT CCA CGC TGT AAA CGA-30 50 -GGA GTG CTT AAC GCG TTA GCT-30 50 -GGT AGT CCA CGC TGT AAA CGA-3’ 50 -GGA GTG CTT AAC GCG TTA GCT-30
TRP19
Ec19-F1 Ec19-R1 Ec19-F2 Ec19-R2
50 -TAA CTC AGG GTG TTA ATT GGT-30 50 -ACA TCA ATA AGC TAC AGG ACT-30 50 -ATT AGT GTT GTG GTT ATG CAA-30 50 -TAC GCT TGC TGA ATA TCA TGA-30
TRP36
Ec36-F1 Ec36-R1 Ec36-F2 Ec36-R2 Ec36-F3 Ec36-R3
50 -AGA TTC TAT GGG ACA TAA TTT GT-30 50 -ACA CAG TAA CAT ATT GCA ATA AG-30 50 -GTA TGT TTC TTT TAT ATC ATG GC-30 50 -GGT TAT ATT TCA GTT ATC AGA AG-30 50 -TGT GTA CAT GGG AAC TCA TCA C-30 50 -TCT TCA GTA ACT CCA CTT GGT-30
a Primers F1 and R1 were used for the first PCR amplification; Primers F2 and R2 were used for the second amplification; Primers F3 and R3 were designed for annealing in the middle of amplicons and used for sequencing towards the 30 and 50 ends, respectively.
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J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32 Table 2 Nucleotide differences in the partial 16S rRNA gene sequence among E. canis isolates available in the GenBank database. Isolates
NGR 64 (JN622141) NGR 80 (JN982337) NGR 94 (JN982339) Brazil CO1 (EF195134) Venezuela VHE (AF373612) Venezuela VDE (AF373613) Greece GR21 (EF011110) Greece GR78 (EF011111) Italy Nero (EU439944) Turkey Kutahya (AY621071) Thailand Bkk07 (EU263991) Thailand Bkk01 (EF139458) TWN 1 (EU106856) TWN 2 (EU123923) TWN 3 (EU143636) TWN 4 (EU143637) Brazil CO2 (EF195135) Japan Kagoshima (AF536827) China Gzh982 (AF162860)b USA Okalahoma (M73221) c USA Florida (M73226)c South America (DQ915970) Israel 611 (U26740) Spain Madrid (AY394465)e USA Jake 2 (CP000107)f
Nucleotide positiona Identity (%)
297
484
758
849
1051
1078
1111
1337
1363
1406
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99.9 99.9 99.9 99.9 99.8 99.8 99.8 99.4 99.9
G A A
G Sd
A G
A C
C T T
A C C
T C
C T
C T
G A
a
Positions based on the sequence of E. canis JN622141 numbering system. The dot () indicates conserved nucleotide. This sequence had a nucleotide deletion at position 947. This sequence had a nucleotide deletion at position 980. d S represents G or C. e This sequence had four nucleotide deletions at positions 126, 129, 614, and 1508, two substitutions at positions 136 and 149 (from A to an uncertain nucleotide), and an uncertain nucleotide insertion between positions 164 and 165. f This sequence had a nucleotide insertion A between positions 973 and 974. b
c
The TRP19 coding sequences (414 bp) amplified from 11 Nigerian isolates were identical to each other. They were all identical to those of the prototype strain from the United States (GenBank accession number DQ858221) and other eight geographically dispersed strains in GenBank, but had three nucleotide differences at positions 9, 323 and 371 from Taiwanese 2–4 isolates (Table 3).
Table 3 Nucleotide differences in the TRP19 gene sequence among canine E. canis isolates available in the GenBank database. Isolates
Nigeria 64 (JN622142) Nigeria 80 (JN982337) Nigeria 94 (JN982340) USA Jake 1 (DQ858221) USA Jake 2 (CP000107) USA Demon (DQ858223) USA Louisiana (DQ858224) USA DJ (DQ858222) USA Florida (DQ858225) Brazil Sao Paulo (DQ860145) Israel Ranana (EU118958) Israel 611 (EU118959) Mexico (DQ858226) TWN 1 (EF527402) TWN 2 (EF560598) TWN 3 (EF587270) TWN 4 (EU139492)
Nucleotide Positionb Identity (%)a
9
71
104
323
371
100 100 100 100 100 100 100 100 100 100 100 99.8 99.8 100 99.3 99.3 99.3
C G G G
G A
A G
A G G G
G A A A
a The values are percentage of nucleotide sequence identities for 414 bp determined from pairwise alignment. b Positions based on the sequence of E. canis JN622142 numbering system. The dot () indicates conserved nucleotide.
The Nigerian isolates also had one nucleotide difference at position 71 and 104 with the Mexico and Israel 611 strains, respectively. The TRP36 coding sequences amplified from 11 Nigerian isolates were identical to each other except for the number of the 27-bp repeat unit in a tandem repeat region, which lies between the 50 end region (429 bp) and the 30 end region (90 bp). Computer-aided analysis with those sequences revealed the presence of three representative genotypes; they were provisionally designated as NGR 64 (n = 4), NGR 80 (n = 5), and NGR 94 (n = 2). The NGR 64 genotype had 8 tandem repeats, the NGR 80 genotype had 18, and the NGR 94 genotype had 12. As shown in Fig. 1, the deduced amino acid sequences of these TRP36 genotypes were compared with those of fifteen published isolates identified from the United States, Brazil, Israel, Cameroon, and Taiwan. It is noteworthy that, without considering the number of tandem repeats, these sequences showed 100% similarity to that of the Cameroon 71 isolate (GenBank accession number DQ146155) previously described by Doyle et al. (2005), but had at least three amino acid changes, in comparison with those from the rest of isolates. Most of the amino acid substitutions are dimorphic, in which only two different amino acids are found at positions wherever substitutions occur (see Fig. 1). As the sequence of TRP36 has greater variation than the sequences of TRP19 and 16S rDNA, a phylogenetic tree of E. canis strains was inferred based on the three representative amino acid sequences of TRP36 obtained in this study and fifteen other sequences of TRP36 from different geographic areas in previous studies by Doyle et al. (2005), Zhang et al. (2008), and Hsieh et al. (2010). In this tree, all of the TRP36 sequences fell into two clusters, the first formed by Taiwanese isolates and the second, by all others (Fig. 2). Notably, the sequences of Nigerian 1–3 and Cameroon 71 isolates formed a separate branch in the second cluster.
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J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32
CAM71 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 NGR64 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 NGR80 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 NGR94 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 Jake1 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 Jake2 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 US-DM US-LS US-OH US-DJ US-FR BRZSP
MLFILMGYCM MLFILMGYCM MLFILMGYCM MLFILMGYCM MLFILMGYCM MLFILMGYCM
LHLTTEITNI LHLTTEITNI LHLTTEITNI LHLTTEITNI LHLTTEITNI LHLTTEITNI
DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ
GERFGVSSGD GERFGVSSGD GERFGVSSGD GERFGVSSGD GERFGVSSGD GERFGVSSGD
LELDIENHPG LELDIENHPG LELDIENHPG LELDVENHPG LELDVENHPG LELDIANHPG
HGYHILFKNN HGYHILFKNN HGYHILFKNN HGYHILFKNN HGYHILFKNN HGYHILFKNN
GHVIS GHVIS GHVIS GHVIS GHVIS GHVIS
065 065 065 065 065 065
IS611 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 ISRNN MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LQLDIENHPG HGYHILFKNN GRVIS 065 TWN 1 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 TWN 2 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 TWN 3 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 TWN 4 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 CAM71 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 NGR64 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 NGR80 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 NGR94 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 Jake1 DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 Jake2 DLHGAKAEDF NFNMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-DM DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-LS DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-OH DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-DJ DLHGAKAEDF NFNMKDHSLN VSFLIDPIAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-FR DLYGAKAEDF NFNMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 BRZSP DLHGVKAEDF NFNMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 IS611 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 ISRNN DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 TWN 1 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 TWN 2 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 TWN 3 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 TWN 4 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 CAM71 IVNQAQVLLP SGV [TEDSVSAPA]16 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY
DQ146155
NGR64 IVNQAQVLLP SGV [TEDSVSAPA]8 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY NGR80 IVNQAQVLLP SGV [TEDSVSAPA]18 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY NGR94 IVNQAQVLLP SGV [TEDSVSAPA]12 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY
JN622143 JN982338 JN982341
Jake1 IVNQAQVLLP SGV [TEDSVSAPA]12 TAAT GSTTSYNHN- TGL--LDLDS DILNMLY Jake2 IVNQAQVLLP SGV [TEDSVSAPA]5 TAAT GSTTSYNHN- TGL--LD--S DILNMLY US-DM IVNQAQVLLP SGV [TEDSVSAPA]16 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY
DQ085427 CP000107 DQ085429
US-LS IVNQAQVLLP SGV [TEDSVSAPA]5 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY US-OH IVNQAQVLLP SGV [TEDSVSAPA]5 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY US-DJ IVNQAQVLLP SGV [TEDSVSAPA]18 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY
DQ146151 DQ085428 DQ146153
US-FR IVNQAQVLLP SGV [TEDSVSAPA]4 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY BRZSP IVNQAQVLLP SGV [TEDSVSAPA]18 TAAT GSTTSYNHN- TELEFLD--S GILNMLY IS611 IVNQAQVLLP SGV [TEDPVSATA]11 TAAT GSTTSYDRNP TGLKFLDLYT QLTL
DQ146152 DQ146154 EF636663
ISRNN IVNQAQVLLP SGV [TEDSVSAPA]10 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY TWN 1 IVSQAQVLLP SGV [TEDSVSAPA]13 TAAT GSTTSYDSD- PGFEFLD--S NILKMLY TWN 2 IVSQAQVLLP SGV [TEDSVSAPA]12 TAAT GSTTSYDSD- TGFEFLD--S NILKMLY
EU118961 EF551366 EF560599
TWN 3 IVSQAQVLLP SGV [TEDSVSAPA]10 TAAT GSTTSYDSD- TGFEFLD--S NILKMLY TWN 4 IVSQAQVLLP SGV [TEDSVSAPA]14 TAAT GSTTSYDSD- TGFEFLD--S NILKMLY
EF651794 EU139491
Fig. 1. Alignment of the deduced amino acid sequence of E. canis TRP36. Amino acids highlighted in grey represent residues divergent from the Cameroon 71 strain sequence, and a dash represents a gap. The nine amino acid sequences in brackets refer to single tandem repeat units. The GenBank accession number for each individual sequence is given at the end of the sequence. Abbreviations of specific E. canis strains: US-DM for the Demon strain (USA), US-LS for the Louisiana strain (USA), US-OK for the Oklahoma strain (USA), US-DJ for the DJ (North Carolina) strain (USA), US-FR for the Florida strain (USA), BRZSP for the Sao Paulo strain (Brazil), ISRNN for the Ranana strain (Israel), IS611 for the 611 strain (Israel), CAM71 for the Cameroon 71 strain, NGR for the Nigerian strain, and TWN for the Taiwanese strain.
J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32
21 44 63
31
E. canis USA-Demon (AAZ40201)
E. canis USA-Jake 1 (AAZ40199) E. canis USA-Okalahoma (AAZ40200) 85 E. canis UAS-Louisiana (ABA39254) E. canis USA-Jake 2 (AAZ68160) 56
E. canis USA-Florida (ABA39255)
51 80 E. canis USA-DJ (ABA39256)
E. canis Brazil-Sao Paulo (ABA39257)
29 17 66
99
E. canis Cameroon-71 (ABA39258) E. canis Nigeria-64 (JN622143)
72 E. canis Nigeria-94 (JN982341) 19 E. canis Nigeria-80 (JN982338)
E. canis Israel-Ranana (ABW91006) E. canis Israel-611 (ABV02078)
E. canis Taiwan-3 (ABV26011) 99
E. canis Taiwan-2 (ABU44524) E. canis Taiwan-4 (ABX71625) 31 34
E. canis Taiwan-1 (ABS82573) E. chaffeensis Arkansas (AAZ40202)
0.1 Fig. 2. Phylogenetic tree based on the E. canis TRP36. Amino acid sequences from geographically dispersed E. canis strains were compared with neighbor-joining method with distance matrix calculation by Kimura’s two parameters, operated by MEGA software version 4, using the orthologous sequence gp47 of Ehrlichia chaffeensis (Doyle et al., 2005) as an outgroup. Scale bar indicates the number of mutations per sequence position. The numbers at the nodes represent the percentage of 1000 bootstrap resamplings. GenBank accession numbers are in parentheses.
4. Discussion Most laboratory diagnosis and monitoring of CME in Nigeria have been based on microscopy in a diagnostic manner. These diagnoses are often ambiguous and may fail to identify the parasite species involved. In the present study, the nearly full-length 16S rRNA genes (1623 bp) of E. canis from 11 infected dogs were amplified and sequenced, providing the first molecular evidence that this infectious agent is involved in canine disease in Nigeria. To date, most molecular epidemiology studies of E. canis have focused on the 16S rDNA, while much less is known about the other genes. However, accumulated data show that molecular characterization of the 16S rDNA has provided little information regarding strain diversity and suggests a high level of conservation (Unver et al., 2003; Aguirre et al., 2004; Siarkou et al., 2007; Vinasco et al., 2007; Matjila et al., 2008; Pinyoowong et al., 2008; Harrus and Waner, 2011; Vargas-Hernández et al., 2011). The 16S rDNA sequences generated in this study also showed a very low diversity when compared with those from other geographically distinct isolates (see Table 2). Nevertheless, our additional analyses of two antigen-encoding genes, TRP19 and TRP36, particularly the latter, did help to identify the genetic variations and phylogeny among E. canis isolates. The TRP36 gene encodes an acidic serine-rich protein that contains a major antibody epitope in the tandem repeat region (McBride et al., 2003). The TRP36 protein has been shown to be one of the first proteins to elicit an E. canis-specific antibody response in the acute phase of the infection (Doyle et al., 2006; Cardenas et al., 2007). Even though it has been reported that N-terminal pre-repeat region (143 amino acids) of TRP36 is highly conserved among eleven E. canis strains from geographically distant areas
including the United States, Brazil, Cameroon, and Israel (Doyle et al., 2005), Hsieh et al. (2010) recently indicated that the same amino acid sequence of TRP36 derived from the Taiwanese isolates is quite different from those of the aforementioned strains, exhibiting 78.3–80.4% identities only. Their findings reinforced the earlier proposition of Zhang et al. (2008) that the TRP36 gene sequence is a good target for evaluation of the genetic diversity of E. canis strains. Using the amino acid sequences of TRP36 generated in this study for sequence alignment, we have demonstrated that except for the number of tandem repeats these sequences bore 100% identity with that of the reported Cameroon 71 strain (see Fig. 1). Additionally, a phylogenetic analysis of TRP36 showed that three representative E. canis Nigerian isolates, together with the Cameroon 71 strain, formed a separate branch in one cluster, which differs distinctly from other branches in the same cluster including those from North America (the United States), South America (Brazil), and West Asia (Israel), as well as another cluster formed by the Taiwanese isolates (see Fig. 2). Considering that Nigeria in West Africa is bordered by Cameroon to the east, it is evident that the phylogenetic tree reveals a geographic correlation between the Nigerian isolates described in this study and the reported Cameroonian isolate, suggesting that they may share a common ancestor. The results presented here also highlight the value of the TRP36 gene as a tool for E. canis genotyping and encourage further investigation for phylogeographic patterns of E. canis strains worldwide using additional samples. In conclusion, the present study shows, for the first time, the presence of E. canis in naturally infected dogs from Nigeria, endorsed by molecular techniques. Molecular evidence indicates the the E. canis genotypes circulating in dogs in Nigeria appear to
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be highly conserved. The results obtained from both sequence alignment and phylogenetic analyses of TRP36 suggest that the E. canis Nigerian isolates are most closely related to the reported Cameroon 71 strain. The significant suggestion of this work is that the TRP36 gene is an ideal genetic marker for genotyping of geographically distributed E. canis strains.
Acknowledgements The authors are grateful to Dr. Goni I. Dogo and Mr. Bitrus Yakubu for their assistance during DNA extraction. We are grateful to the management of National Veterinary Research Institute, Vom, Nigeria for logistic support and permission to conduct and publish this work. This work was supported in part by the Taiwanese government through the research grant to National Chung Hsing University (NCHU-CC98116).
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Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria Joshua Kamani a, Chung-Chan Lee b, Ayuba M. Haruna c, Ping-Jun Chung b, Paul R. Weka a, Yang-Tsung Chung b,⇑ a
Parasitology Division, National Veterinary Research Institute PMB 01, Vom, Plateau State, Nigeria Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan c State Veterinary Hospital Jos, Ministry of Agriculture and Natural Resources, Plateau State, Nigeria b
a r t i c l e
i n f o
Article history: Received 28 March 2012 Accepted 28 July 2012
Keywords: Ehrlichia canis Dogs PCR Phylogenetic analysis TRP36 gene Nigeria
a b s t r a c t The present study aimed to detect the presence of Ehrlichia canis in naturally infected dogs in Nigeria, using a combination of PCR and sequence analysis of the 16S rRNA gene and two genes encoding the tandem repeat-containing proteins (TRPs), TRP19 and TRP36. Out of a total of 100 blood samples collected from domestic dogs presented to veterinary hospitals in Jos, the capital city of Plateau State of Nigeria, 11 were positive in nested PCR for E. canis. Sequencing results for these amplicons showed that all of the 16S rDNA sequences (1623 bp) or the TRP19 coding sequences (414 bp) were identical to each other and had very high similarities (99.3–100%) with those from other E. canis strains accessible in GenBank. The TRP36 gene sequences derived from the 11 Nigerian isolates were identical to each other except for the number of the 27-bp repeat unit in a tandem repeat region, which was found to be 8, 12 or 18. Without considering the number of tandem repeats, these sequences had 100% identity to that of the reported Cameroon 71 isolate, but distinctly differed from those obtained from other geographically distant E. canis strains previously published. A phylogenetic tree of E. canis based on the TRP36 amino acid sequences showed that the Nigerian isolates and the Cameroon 71 isolate fell into a separate clade, indicating that they may share a common ancestor. Overall, this study not only provides the first molecular evidence of E. canis infections in dogs from Nigeria but also highlights the value of the TRP36 gene as a tool to classify E. canis isolates and to elucidate their phylogeographic relationships. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Canine monocytic ehrlichiosis (CME) caused by Ehrlichia canis is of veterinary importance worldwide. The causative agent of the disease is a Gram negative, obligatory intracellular bacterium, which is enveloped with a rippled thin outer membrane (Rikihisa et al., 1997; Boozer and Macintire, 2003). E. canis has special tropism for monocytes and macrophages (Huxsoll et al., 1970). It consists of a single circular chromosome containing 1,315,030 nucleotides (Mavromatis et al., 2006). The agent is predominantly transmitted by the brown dog tick Rhipicephalus sanguineus (Dantas-Torres, 2008; Stich et al., 2008). CME has been reported throughout the world, with a higher frequency in tropical and subtropical regions (Vinasco et al., 2007; Rar and Golovljova, 2011). Diagnosis of the disease can be challenging due to its different phases and multiple clinical manifestations (Harrus and Waner, 2011). Traditional diagnostic techniques such ⇑ Corresponding author. Address: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. Tel.: +886 4 2284 0751; fax: +886 4 2286 2073. E-mail address: [email protected] (Y.-T. Chung). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.07.031
as hematology, cytology, serology and isolation are valuable diagnostic tools for CME. However, such methods are labor-intensive and the results obtained are often ambiguous because of their low sensitivities or inability to differentiate past exposure to the disease or active infections (Breitschwerdt et al., 1998). The definitive diagnosis of E. canis infection therefore requires molecular techniques (Harrus et al., 1998; Harrus and Waner, 2011) especially the polymerase chain reaction (PCR), which has been used extensively for specific diagnosis of tick-borne protozoal and ehrlichial infections in dogs in developed countries (Gal et al., 2008). This is not the case with most developing countries due to decay in infrastructure and lack of expertise to conduct such tests. In Nigeria, the diagnosis of infections with Ehrlichia species is usually based on the detection of pathogens in peripheral blood under a microscope. The search for morulae by microscopy is difficult, time-consuming and has been estimated to be successful in only about 4% of cases (Woody and Hoskins, 1991). Such challenges in the diagnosis of ehrlichiosis in dogs in Nigeria have led to wrong conclusion among veterinarians that the disease rarely occur, and thus, is not considered a major problem. As such there has not been any published report on molecular status of CME agent in Nigeria in the last two decades with no information
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J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32
available on its DNA sequence and phylogeny. The purpose of this study was thus to assess the presence of E. canis in dogs in Nigeria using molecular methods. We sought to amplify with PCR the 16S ribosomal RNA gene (16S rDNA) and two genes encoding the tandem repeat-containing proteins (TRPs), TRP19 (formerly gp19) and TRP36 (gp36), characterized by previous studies (Doyle et al., 2005; Hsieh et al., 2010) followed by sequencing of those fragments. We also conducted sequence alignment and phylogenetic analysis to identify E. canis genotypes. 2. Materials and methods
clones were picked and purified individually using the QIAprep Spin Miniprep Kit (Qiagen), and then sequenced by using an ABI PRISM 3730 capillary sequencer (Applied Biosystems) and the Dye Terminator Cycler Sequencing Kit (Applied Biosystems) with a vector primer (T7 or SP6) and an appropriate internal sequencing primer (see Table 1). Both the sense and antisense strands of each PCR amplicon were sequenced. Sequences were manually edited to resolve ambiguities. A consensus sequence was obtained for each amplicon by comparing both the sense and antisense sequences. Representative nucleotide sequences for the E. canis genes reported here have been deposited in the GenBank database under accession numbers JN622141–3 and JN982336–41.
2.1. Collection of blood samples and DNA extraction 2.3. Sequence and phylogenetic analyses Blood was collected from 100 dogs manifesting clinical signs of tick-borne diseases that were referred to veterinary hospitals in Jos, the capital city of Plateau State of Nigeria, from February to July 2011. Blood was collected from cephalic vein into sterile tubes with anticoagulant (EDTA), and held at 4 °C until arrival at the laboratory for further processing. Genomic DNA was extracted from 200 ll of whole blood samples by using AxyPrep TM Genomic DNA extraction kit (Axygen Biosciences, Axygen Scientific, Inc. USA) according to manufacturer’s recommendation. Nucleic acid was eluted into 100 ll of elution buffer and stored at 20 °C until further use. 2.2. PCR amplification and sequencing Primers used for the amplification and sequencing of the 16S rRNA, TRP19 and TRP36 genes of E. canis (Cardenas et al., 2007; McBride et al., 2007) were designed using primer design software (Primer Select, DNASTAR, Madison, WI, USA) and E. canis genome sequence information (Mavromatis et al., 2006) and are presented in Table 1. PCR amplification was carried out as described previously (Hsieh et al., 2010). The resulting PCR products were electrophoresed on a 1.2% agarose gel stained with ethidium bromide to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 kb Plus DNA Ladder, Promega). In each case, the single amplicon of the expected size was column purified using the QIAquick PCR Purification Kit (Qiagen) and ligated into the pGEM-T vector (Promega) for subsequent transformation in the Escherichia coli DH5a competent cells. For each PCR amplicon, at least three recombinant
The BLAST program (http://www.ncbi.nlm.nih.gov/ BLAST) was used for the comparison and the analysis of sequence data obtained in this study with those previously deposited in GenBank. Multiple sequence alignment was conducted using the program AlignX (Vector NTI Suite V5.5, InforMax, North Bethesda, MD, USA) with an engine based on the Clustal W algorithm (Thompson et al., 1994). A TRP36 phylogenetic tree was inferred using the neighbor-joining method as implemented by MEGA software version 4 (Tamura et al., 2004). The distance matrix of amino acid divergences was calculated according to Kimura’s two parameter model furnished by MEGA. A bootstrap resampling technique of 1000 replications was conducted to statistically support the reliabilities of the nodes on the tree. 3. Results All blood samples were subjected to DNA extraction followed by nested PCR amplification with the 16S rDNA-targeted primers (see Table 1). Among 100 samples examined, 11 (11%) were positive for E. canis. Sequencing results for those amplicons showed that all 16S rDNA sequences of 1623 base pairs (bp) derived from 11 Nigerian isolates were identical to each other and bore 99.4– 100% identities with the corresponding sequences from E. canis strains in other geographic areas (Table 2). This is in agreement with previous investigations (Aguirre et al., 2004; Siarkou et al., 2007; Yu et al., 2007; Pinyoowong et al., 2008), indicating that the genetic profile of canine E. canis strains based on the 16S rDNA sequence was highly conserved.
Table 1 Primers used for the amplification and sequencing of the 16S rRNA, TRP19 and TRP36 genes from the Nigerian E. canis isolates. Target
Primera
Sequence
16S rDNA
Ec16S-F1 Ec16S-R1 Ec16S-F2 Ec16S-R2 Ec16S-F3 Ec16S-R3
50 -TCA ACC CAT GGC TAA ATG TCA-30 50 -CAG ATG TGG AGA TAA AGG CCT-30 50 -GGT AGT CCA CGC TGT AAA CGA-30 50 -GGA GTG CTT AAC GCG TTA GCT-30 50 -GGT AGT CCA CGC TGT AAA CGA-3’ 50 -GGA GTG CTT AAC GCG TTA GCT-30
TRP19
Ec19-F1 Ec19-R1 Ec19-F2 Ec19-R2
50 -TAA CTC AGG GTG TTA ATT GGT-30 50 -ACA TCA ATA AGC TAC AGG ACT-30 50 -ATT AGT GTT GTG GTT ATG CAA-30 50 -TAC GCT TGC TGA ATA TCA TGA-30
TRP36
Ec36-F1 Ec36-R1 Ec36-F2 Ec36-R2 Ec36-F3 Ec36-R3
50 -AGA TTC TAT GGG ACA TAA TTT GT-30 50 -ACA CAG TAA CAT ATT GCA ATA AG-30 50 -GTA TGT TTC TTT TAT ATC ATG GC-30 50 -GGT TAT ATT TCA GTT ATC AGA AG-30 50 -TGT GTA CAT GGG AAC TCA TCA C-30 50 -TCT TCA GTA ACT CCA CTT GGT-30
a Primers F1 and R1 were used for the first PCR amplification; Primers F2 and R2 were used for the second amplification; Primers F3 and R3 were designed for annealing in the middle of amplicons and used for sequencing towards the 30 and 50 ends, respectively.
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J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32 Table 2 Nucleotide differences in the partial 16S rRNA gene sequence among E. canis isolates available in the GenBank database. Isolates
NGR 64 (JN622141) NGR 80 (JN982337) NGR 94 (JN982339) Brazil CO1 (EF195134) Venezuela VHE (AF373612) Venezuela VDE (AF373613) Greece GR21 (EF011110) Greece GR78 (EF011111) Italy Nero (EU439944) Turkey Kutahya (AY621071) Thailand Bkk07 (EU263991) Thailand Bkk01 (EF139458) TWN 1 (EU106856) TWN 2 (EU123923) TWN 3 (EU143636) TWN 4 (EU143637) Brazil CO2 (EF195135) Japan Kagoshima (AF536827) China Gzh982 (AF162860)b USA Okalahoma (M73221) c USA Florida (M73226)c South America (DQ915970) Israel 611 (U26740) Spain Madrid (AY394465)e USA Jake 2 (CP000107)f
Nucleotide positiona Identity (%)
297
484
758
849
1051
1078
1111
1337
1363
1406
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99.9 99.9 99.9 99.9 99.8 99.8 99.8 99.4 99.9
G A A
G Sd
A G
A C
C T T
A C C
T C
C T
C T
G A
a
Positions based on the sequence of E. canis JN622141 numbering system. The dot () indicates conserved nucleotide. This sequence had a nucleotide deletion at position 947. This sequence had a nucleotide deletion at position 980. d S represents G or C. e This sequence had four nucleotide deletions at positions 126, 129, 614, and 1508, two substitutions at positions 136 and 149 (from A to an uncertain nucleotide), and an uncertain nucleotide insertion between positions 164 and 165. f This sequence had a nucleotide insertion A between positions 973 and 974. b
c
The TRP19 coding sequences (414 bp) amplified from 11 Nigerian isolates were identical to each other. They were all identical to those of the prototype strain from the United States (GenBank accession number DQ858221) and other eight geographically dispersed strains in GenBank, but had three nucleotide differences at positions 9, 323 and 371 from Taiwanese 2–4 isolates (Table 3).
Table 3 Nucleotide differences in the TRP19 gene sequence among canine E. canis isolates available in the GenBank database. Isolates
Nigeria 64 (JN622142) Nigeria 80 (JN982337) Nigeria 94 (JN982340) USA Jake 1 (DQ858221) USA Jake 2 (CP000107) USA Demon (DQ858223) USA Louisiana (DQ858224) USA DJ (DQ858222) USA Florida (DQ858225) Brazil Sao Paulo (DQ860145) Israel Ranana (EU118958) Israel 611 (EU118959) Mexico (DQ858226) TWN 1 (EF527402) TWN 2 (EF560598) TWN 3 (EF587270) TWN 4 (EU139492)
Nucleotide Positionb Identity (%)a
9
71
104
323
371
100 100 100 100 100 100 100 100 100 100 100 99.8 99.8 100 99.3 99.3 99.3
C G G G
G A
A G
A G G G
G A A A
a The values are percentage of nucleotide sequence identities for 414 bp determined from pairwise alignment. b Positions based on the sequence of E. canis JN622142 numbering system. The dot () indicates conserved nucleotide.
The Nigerian isolates also had one nucleotide difference at position 71 and 104 with the Mexico and Israel 611 strains, respectively. The TRP36 coding sequences amplified from 11 Nigerian isolates were identical to each other except for the number of the 27-bp repeat unit in a tandem repeat region, which lies between the 50 end region (429 bp) and the 30 end region (90 bp). Computer-aided analysis with those sequences revealed the presence of three representative genotypes; they were provisionally designated as NGR 64 (n = 4), NGR 80 (n = 5), and NGR 94 (n = 2). The NGR 64 genotype had 8 tandem repeats, the NGR 80 genotype had 18, and the NGR 94 genotype had 12. As shown in Fig. 1, the deduced amino acid sequences of these TRP36 genotypes were compared with those of fifteen published isolates identified from the United States, Brazil, Israel, Cameroon, and Taiwan. It is noteworthy that, without considering the number of tandem repeats, these sequences showed 100% similarity to that of the Cameroon 71 isolate (GenBank accession number DQ146155) previously described by Doyle et al. (2005), but had at least three amino acid changes, in comparison with those from the rest of isolates. Most of the amino acid substitutions are dimorphic, in which only two different amino acids are found at positions wherever substitutions occur (see Fig. 1). As the sequence of TRP36 has greater variation than the sequences of TRP19 and 16S rDNA, a phylogenetic tree of E. canis strains was inferred based on the three representative amino acid sequences of TRP36 obtained in this study and fifteen other sequences of TRP36 from different geographic areas in previous studies by Doyle et al. (2005), Zhang et al. (2008), and Hsieh et al. (2010). In this tree, all of the TRP36 sequences fell into two clusters, the first formed by Taiwanese isolates and the second, by all others (Fig. 2). Notably, the sequences of Nigerian 1–3 and Cameroon 71 isolates formed a separate branch in the second cluster.
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J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32
CAM71 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 NGR64 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 NGR80 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 NGR94 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 Jake1 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 Jake2 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 US-DM US-LS US-OH US-DJ US-FR BRZSP
MLFILMGYCM MLFILMGYCM MLFILMGYCM MLFILMGYCM MLFILMGYCM MLFILMGYCM
LHLTTEITNI LHLTTEITNI LHLTTEITNI LHLTTEITNI LHLTTEITNI LHLTTEITNI
DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ DFAHDFHIHQ
GERFGVSSGD GERFGVSSGD GERFGVSSGD GERFGVSSGD GERFGVSSGD GERFGVSSGD
LELDIENHPG LELDIENHPG LELDIENHPG LELDVENHPG LELDVENHPG LELDIANHPG
HGYHILFKNN HGYHILFKNN HGYHILFKNN HGYHILFKNN HGYHILFKNN HGYHILFKNN
GHVIS GHVIS GHVIS GHVIS GHVIS GHVIS
065 065 065 065 065 065
IS611 MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LELDIENHPG HGYHILFKNN GHVIS 065 ISRNN MLFILMGYCM LHLTTEITNI DFAHDFHIHQ GERFGVSSGD LQLDIENHPG HGYHILFKNN GRVIS 065 TWN 1 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 TWN 2 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 TWN 3 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 TWN 4 MLFILMGYCM LHLTTEITGI DFSNDFHIHS GERFVVVSGD IQLEVGNSGE HGYHILFKNG GHVIS 065 CAM71 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 NGR64 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 NGR80 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 NGR94 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 Jake1 DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 Jake2 DLHGAKAEDF NFNMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-DM DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-LS DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-OH DLHGAKAEDF NFDMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-DJ DLHGAKAEDF NFNMKDHSLN VSFLIDPIAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 US-FR DLYGAKAEDF NFNMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISVHAYQ DGCDNCVHGN PSRPA 130 BRZSP DLHGVKAEDF NFNMKDHSLN VSFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 IS611 DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 ISRNN DLHGVKAEDF NFNMKDHSLN ASFLIDPMAP FHELDVNNHP NFFISMHAYQ DGCDNCVHGN PSRPA 130 TWN 1 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 TWN 2 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 TWN 3 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 TWN 4 DLHGVKAEYF NFDMKDRSLN ASFLIDPMAP FHELDVKNHP NFSISMHAEG D-CDNCVHGN SSLLP 129 CAM71 IVNQAQVLLP SGV [TEDSVSAPA]16 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY
DQ146155
NGR64 IVNQAQVLLP SGV [TEDSVSAPA]8 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY NGR80 IVNQAQVLLP SGV [TEDSVSAPA]18 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY NGR94 IVNQAQVLLP SGV [TEDSVSAPA]12 TAAT GSTTSYNHN- TGLEFLDLGS DILNMLY
JN622143 JN982338 JN982341
Jake1 IVNQAQVLLP SGV [TEDSVSAPA]12 TAAT GSTTSYNHN- TGL--LDLDS DILNMLY Jake2 IVNQAQVLLP SGV [TEDSVSAPA]5 TAAT GSTTSYNHN- TGL--LD--S DILNMLY US-DM IVNQAQVLLP SGV [TEDSVSAPA]16 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY
DQ085427 CP000107 DQ085429
US-LS IVNQAQVLLP SGV [TEDSVSAPA]5 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY US-OH IVNQAQVLLP SGV [TEDSVSAPA]5 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY US-DJ IVNQAQVLLP SGV [TEDSVSAPA]18 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY
DQ146151 DQ085428 DQ146153
US-FR IVNQAQVLLP SGV [TEDSVSAPA]4 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY BRZSP IVNQAQVLLP SGV [TEDSVSAPA]18 TAAT GSTTSYNHN- TELEFLD--S GILNMLY IS611 IVNQAQVLLP SGV [TEDPVSATA]11 TAAT GSTTSYDRNP TGLKFLDLYT QLTL
DQ146152 DQ146154 EF636663
ISRNN IVNQAQVLLP SGV [TEDSVSAPA]10 TAAT GSTTSYNHN- TGLEFLDLDS DILNMLY TWN 1 IVSQAQVLLP SGV [TEDSVSAPA]13 TAAT GSTTSYDSD- PGFEFLD--S NILKMLY TWN 2 IVSQAQVLLP SGV [TEDSVSAPA]12 TAAT GSTTSYDSD- TGFEFLD--S NILKMLY
EU118961 EF551366 EF560599
TWN 3 IVSQAQVLLP SGV [TEDSVSAPA]10 TAAT GSTTSYDSD- TGFEFLD--S NILKMLY TWN 4 IVSQAQVLLP SGV [TEDSVSAPA]14 TAAT GSTTSYDSD- TGFEFLD--S NILKMLY
EF651794 EU139491
Fig. 1. Alignment of the deduced amino acid sequence of E. canis TRP36. Amino acids highlighted in grey represent residues divergent from the Cameroon 71 strain sequence, and a dash represents a gap. The nine amino acid sequences in brackets refer to single tandem repeat units. The GenBank accession number for each individual sequence is given at the end of the sequence. Abbreviations of specific E. canis strains: US-DM for the Demon strain (USA), US-LS for the Louisiana strain (USA), US-OK for the Oklahoma strain (USA), US-DJ for the DJ (North Carolina) strain (USA), US-FR for the Florida strain (USA), BRZSP for the Sao Paulo strain (Brazil), ISRNN for the Ranana strain (Israel), IS611 for the 611 strain (Israel), CAM71 for the Cameroon 71 strain, NGR for the Nigerian strain, and TWN for the Taiwanese strain.
J. Kamani et al. / Research in Veterinary Science 94 (2013) 27–32
21 44 63
31
E. canis USA-Demon (AAZ40201)
E. canis USA-Jake 1 (AAZ40199) E. canis USA-Okalahoma (AAZ40200) 85 E. canis UAS-Louisiana (ABA39254) E. canis USA-Jake 2 (AAZ68160) 56
E. canis USA-Florida (ABA39255)
51 80 E. canis USA-DJ (ABA39256)
E. canis Brazil-Sao Paulo (ABA39257)
29 17 66
99
E. canis Cameroon-71 (ABA39258) E. canis Nigeria-64 (JN622143)
72 E. canis Nigeria-94 (JN982341) 19 E. canis Nigeria-80 (JN982338)
E. canis Israel-Ranana (ABW91006) E. canis Israel-611 (ABV02078)
E. canis Taiwan-3 (ABV26011) 99
E. canis Taiwan-2 (ABU44524) E. canis Taiwan-4 (ABX71625) 31 34
E. canis Taiwan-1 (ABS82573) E. chaffeensis Arkansas (AAZ40202)
0.1 Fig. 2. Phylogenetic tree based on the E. canis TRP36. Amino acid sequences from geographically dispersed E. canis strains were compared with neighbor-joining method with distance matrix calculation by Kimura’s two parameters, operated by MEGA software version 4, using the orthologous sequence gp47 of Ehrlichia chaffeensis (Doyle et al., 2005) as an outgroup. Scale bar indicates the number of mutations per sequence position. The numbers at the nodes represent the percentage of 1000 bootstrap resamplings. GenBank accession numbers are in parentheses.
4. Discussion Most laboratory diagnosis and monitoring of CME in Nigeria have been based on microscopy in a diagnostic manner. These diagnoses are often ambiguous and may fail to identify the parasite species involved. In the present study, the nearly full-length 16S rRNA genes (1623 bp) of E. canis from 11 infected dogs were amplified and sequenced, providing the first molecular evidence that this infectious agent is involved in canine disease in Nigeria. To date, most molecular epidemiology studies of E. canis have focused on the 16S rDNA, while much less is known about the other genes. However, accumulated data show that molecular characterization of the 16S rDNA has provided little information regarding strain diversity and suggests a high level of conservation (Unver et al., 2003; Aguirre et al., 2004; Siarkou et al., 2007; Vinasco et al., 2007; Matjila et al., 2008; Pinyoowong et al., 2008; Harrus and Waner, 2011; Vargas-Hernández et al., 2011). The 16S rDNA sequences generated in this study also showed a very low diversity when compared with those from other geographically distinct isolates (see Table 2). Nevertheless, our additional analyses of two antigen-encoding genes, TRP19 and TRP36, particularly the latter, did help to identify the genetic variations and phylogeny among E. canis isolates. The TRP36 gene encodes an acidic serine-rich protein that contains a major antibody epitope in the tandem repeat region (McBride et al., 2003). The TRP36 protein has been shown to be one of the first proteins to elicit an E. canis-specific antibody response in the acute phase of the infection (Doyle et al., 2006; Cardenas et al., 2007). Even though it has been reported that N-terminal pre-repeat region (143 amino acids) of TRP36 is highly conserved among eleven E. canis strains from geographically distant areas
including the United States, Brazil, Cameroon, and Israel (Doyle et al., 2005), Hsieh et al. (2010) recently indicated that the same amino acid sequence of TRP36 derived from the Taiwanese isolates is quite different from those of the aforementioned strains, exhibiting 78.3–80.4% identities only. Their findings reinforced the earlier proposition of Zhang et al. (2008) that the TRP36 gene sequence is a good target for evaluation of the genetic diversity of E. canis strains. Using the amino acid sequences of TRP36 generated in this study for sequence alignment, we have demonstrated that except for the number of tandem repeats these sequences bore 100% identity with that of the reported Cameroon 71 strain (see Fig. 1). Additionally, a phylogenetic analysis of TRP36 showed that three representative E. canis Nigerian isolates, together with the Cameroon 71 strain, formed a separate branch in one cluster, which differs distinctly from other branches in the same cluster including those from North America (the United States), South America (Brazil), and West Asia (Israel), as well as another cluster formed by the Taiwanese isolates (see Fig. 2). Considering that Nigeria in West Africa is bordered by Cameroon to the east, it is evident that the phylogenetic tree reveals a geographic correlation between the Nigerian isolates described in this study and the reported Cameroonian isolate, suggesting that they may share a common ancestor. The results presented here also highlight the value of the TRP36 gene as a tool for E. canis genotyping and encourage further investigation for phylogeographic patterns of E. canis strains worldwide using additional samples. In conclusion, the present study shows, for the first time, the presence of E. canis in naturally infected dogs from Nigeria, endorsed by molecular techniques. Molecular evidence indicates the the E. canis genotypes circulating in dogs in Nigeria appear to
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be highly conserved. The results obtained from both sequence alignment and phylogenetic analyses of TRP36 suggest that the E. canis Nigerian isolates are most closely related to the reported Cameroon 71 strain. The significant suggestion of this work is that the TRP36 gene is an ideal genetic marker for genotyping of geographically distributed E. canis strains.
Acknowledgements The authors are grateful to Dr. Goni I. Dogo and Mr. Bitrus Yakubu for their assistance during DNA extraction. We are grateful to the management of National Veterinary Research Institute, Vom, Nigeria for logistic support and permission to conduct and publish this work. This work was supported in part by the Taiwanese government through the research grant to National Chung Hsing University (NCHU-CC98116).
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