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A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil
Veterinary Parasitology 143 (2007) 189–195 www.elsevier.com/locate/vetpar
Short communication
A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil Marcelo B. Labruna a,b,*, Jere W. McBride a, Luis Marcelo A. Camargo c, Daniel M. Aguiar b, Michael J. Yabsley d, William R. Davidson d, Ellen Y. Stromdahl e, Phillip C. Williamson f, Roger W. Stich g, S. Wesley Long a, Erney P. Camargo c, David H. Walker a a
Department of Pathology, University of Texas Medical Branch, Galveston, TX 7755-0609, USA Departamento de Medicina Veterinaria Preventiva e Saude Animal, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil c Departamento de Parasitologia, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil d Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA e U.S. Army Center for Health Promotion and Preventive Medicine, APG, MD 21010, USA f DNA/Identity Laboratory, Department of Pathology and Anatomy, University of North Texas Health Science Center, Ft. Worth, TX 76107-2699, USA g Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210, USA b
Received 8 February 2006; received in revised form 1 July 2006; accepted 1 August 2006
Abstract A molecular epidemiologic investigation in two Brazilian states (Rondoˆnia and Sa˜o Paulo) was undertaken to determine if Ehrlichia species responsible for human and animal ehrlichioses in North America could be found in Brazilian vectors, potential natural mammalian reservoirs and febrile human patients with a tick bite history. Samples, including 376 ticks comprising 9 Amblyomma species, 29 capybara (Hydrochaeris hydrochaeris) spleens, 5 canine blood, and 75 human blood samples from febrile patients with history of tick bites were tested by a real-time PCR assay targeting a fragment of the Ehrlichia dsb gene. Ehrlichia DNA was not detected in any tick, capybara or human samples. In contrast, 4 out of 5 dogs contained Ehrlichia canis DNA in their blood, which were sequenced, representing the first report of E. canis infecting dogs in the Amazon region of Brazil. Further studies are needed to evaluate the presence of other agents of human and animal ehrlichioses in Brazil. # 2006 Elsevier B.V. All rights reserved. Keywords: Ehrlichia; Dsb gene; Real-time PCR; Ticks; Brazil
1. Introduction
* Corresponding author at: Departamento de Medicina Veterinaria Preventiva e Saude Animal, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitaria, Sa˜o Paulo, SP 05508-270, Brazil. Tel.: +55 11 3091 1394; fax: +55 11 3091 7928. E-mail address: [email protected] (M.B. Labruna). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.08.005
The genus Ehrlichia currently consists of five recognized species, which include E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium (Dumler et al., 2001). Other potential species, yet to be established in tissue culture, have been identified by molecular methods (Shibata et al., 2000; Wen et al., 2002; Inayoshi et al.,
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2004). All ehrlichial species infect vertebrate hosts and are transmitted by ticks. E. chaffeensis and E. ewingii are important agents of human disease in North America, whereas E. canis, E. ruminantium and E. ewingii are pathogens of major veterinary importance (Dumler et al., 2001). The discovery and emergence of E. chaffeensis as a human pathogen in the United States (Anderson et al., 1991; Paddock and Childs, 2003), and the improvements in molecular technologies during the last two decades has created an enormous interest in the global epidemiology and ecology of human ehrlichioses. Consequently, numerous studies investigating the ecological aspects of human ehrlichiosis in the United States have been reported (Paddock and Childs, 2003) and several new Ehrlichia genotypes found to infect ticks and animals have been reported outside the United States (Cao et al., 2000; Allsopp and Allsopp, 2001; Jenkins et al., 2001; Wen et al., 2002; Inayoshi et al., 2004). Currently, the only Ehrlichia species recognized in South America is E. canis, which is the primary etiologic agent of canine monocytic ehrlichiosis (CME) (Dumler et al., 2001; Moreira et al., 2003). In southern Brazil, CME is one of the most important infectious diseases of dogs (Dagnone et al., 2003; Moreira et al., 2003). Recent reports indicated that E. canis is a zoonotic agent causing asymptomatic or symptomatic infection in humans from Venezuela (Perez et al., 1996, 2005). In addition, there is serological evidence for human ehrlichiosis in Argentina and Brazil (Ripoli et al., 1999; Calic et al., 2004). Previously, no epidemiological study on ehrlichiosis (other than E. canis in urban areas) has been performed in Brazil. Thus, our study was a preliminary investigation, which tested the presence of ehrlichial DNA in tick, human, and animal samples from rural areas collected during other independent studies performed in Brazil (Labruna et al., 2004a,b, 2005). For this purpose, we used a highly sensitive and specific quantitative real-time PCR assay targeting the dsb gene of Ehrlichia species. 2. Materials and methods 2.1. Clinical samples from Brazil A total of 376 ticks were tested individually (n = 154) or in pools containing 2–10 ticks. The following tick species collected in the Amazon forest (Monte Negro and Gov. Jorge Teixeira Counties) in the state of Rondoˆnia (number of specimens in parenthesis) were tested: Amblyomma ovale (n = 121), Amblyomma cajennense (n = 41), Amblyomma scalpturatum (n = 35), Amblyomma naponense (n = 36), Amblyomma
oblongoguttatum (n = 30), Amblyomma coelebs (n = 10), and Amblyomma humerale (n = 6). The following tick species collected in the state of Sa˜o Paulo (number of specimens in parenthesis) were tested: Amblyomma dubitatum (=Amblyomma cooperi) (n = 32) and A. cajennense (n = 35) from Pedreira County; Amblyomma triste (n = 30) from Paulice´ia County. All ticks were collected as free-living in the vegetation except for A. humerale, which were collected from a tortoise during a previous study (Labruna et al., 2005). Capybara (Hydrocaheris hydrochaeris) spleens (n = 29) from Piracicaba County, state of Sa˜o Paulo, human blood samples (n = 75) from the rural area of Monte Negro County, state of Rondoˆnia, and canine blood samples (n = 5) from the urban area of Monte Negro were also tested. The human samples were collected from febrile patients with history of a tick exposure, prior to administration of antibiotic therapy. The canine samples were collected from domestic dogs showing infestation by Rhipicephalus sanguineus ticks and clinical signs compatible with CME (apathy, pale mucous membranes, and epistaxis). DNA from tick (whole tick body), capybara (10 mg of each spleen), canine, and human samples (100 ml of whole blood from each sample) was extracted using the DNeasy Tissue Kit (Qiagen, Chatsworth, CA) following the manufacturer’s protocols. 2.2. Testing Brazilian samples by real-time PCR assay All collected samples were tested by a real-time PCR assay as follows. Primers were designed based on the alignment of the 738-bp of the disulfide bond formation protein gene (dsb) of five Ehrlichia spp. available in GenBank (Table 1) using the CLUSTAL algorithm of the program MegAlign (DNAstar, Lasergene, Madison, WI). E. ewingii was the only species not included in the alignment because its dsb sequence was still undetermined. To design genus specific primers that could be used to amplify the dsb gene of all Ehrlichia species, we identified regions within the gene that were highly conserved among the different species. The nucleic acid sequence identities between the ehrlichial dsb genes varied from 74.6 to 91.8%, but two highly conserved regions were found in the gene alignment. A pair of primers designated as Dsb-330 and Dsb-728, were manually designed to correspond to these two conserved regions and amplify a 409-bp fragment of the Ehrlichia dsb (Table 1) (Doyle et al., 2005). To improve primer specificity (both primers have melting temperature >58 8C) and to minimize formation of primer dimers,
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Table 1 Nucleotide sequence alignment for the regions of the ehrlichial dsb gene corresponding to primers Dsb-330 and Dsb-728
the consensus primers were modified by arbitrary single nucleic acid substitutions (Dsb 330 position 1, A–G; Dsb 728 position 7, A–G) in the 50 region. Real-time PCR reactions were performed using a BioRad i-Cycler, with SYBR I green as the fluorescent dye. The amplification reaction, in a final volume of 50 ml, contained 25 ml of iQTMSYBR1Green I Supermix (Bio-Rad, Hercules, CA), 1 ml of each primer at 20 mM (final concentration of 400 nM), 18 ml of molecular grade water, and 5 ml of template containing 100–300 ng DNA. Primer concentrations were optimized in previous assays by spanning different initial concentrations of oligonucleotides. Real-time PCR cycling conditions were 1 cycle at 95 8C for 2 min followed by 50 cycles of 15 s at 95 8C, 30 s at 58 8C and 30 s at 72 8C, followed by an amplicon melt curve analysis. In each set of reactions, a positive control (E. chaffeensis DNA) and three negative water controls were included. 2.3. Testing the DNA extraction protocol To validate the DNA extraction protocol used for our Brazilian samples, blood samples from three E. chaffeensis-infected white-tail deer (provided by M.J.Y.), one blood sample from an experimentally E. canis-infected dog (provided by R.W.S.), and an experimentally E. canis-infected pool of two Rhipicephalus sanguineus ticks (provided by M.B.L.), all from the United States, were subjected to the same DNA extraction protocol cited above. These DNA samples were tested by the real-time PCR assay described above. 2.4. Testing the real-time PCR assay To validate the real-time PCR assay used for the Brazilian samples, the following DNA samples from
animals and ticks, previously shown by nested 16S rRNA PCR to contain ehrlichial DNA (Stromdahl et al., 2001; Yabsley et al., 2003; Long et al., 2004), were tested by the real-time PCR: DNA from peripheral blood of five white-tailed deer naturally infected with E. chaffeensis, one A. americanum tick naturally infected with E. ewingii, and five A. americanum ticks naturally infected with E. chaffeensis. 2.5. Testing the sensitivity and specificity of the real-time PCR assay To evaluate the sensitivity of the assay for detection of Ehrlichia spp. DNA, the entire dsb gene of E. chaffeensis (738 bp) was amplified as previously described (McBride et al., 2002) and cloned into a plasmid (pCR1T7 NT-TOPO1 TA Cloning Kit, Invitrogen, Carlsbad, CA). The DNA concentration of the purified plasmid was determined using a spectrophotometer at A260 (Perkin-Elmer MBA 2000, Norwalk, CT), and the number of plasmid copies (equivalent to the number of E. chaffeensis dsb gene copies) was calculated. Serial 10fold dilutions from 106 to 100 of plasmid containing the E. chaffeensis dsb gene were prepared in diluent containing DNA extracted from uninfected white-tailed deer blood. The plasmid dilutions mixed with 200 ng of deer DNA were used as standards in the real-time PCR assay and tested in duplicate. The same procedure described above was performed with the dsb gene of E. canis, except that uninfected R. sanguineus tick DNA was used as diluent, giving a final amount of 300 ng of tick DNA per standard template. Both DNA extractions of R. sanguineus ticks and deer blood were performed using the DNeasy Tissue Kit as cited above. The specificity of the real-time PCR assay was determined by testing genomic DNA of tick, capybara,
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Table 2 Samples tested to validate the real-time PCR assay DNA template sourcea
Infection status
Spiked with E. chaffeensis DNA b
Ct values
DH82 cell culture DH82 cell culture P388D1 cell culture Murine spleen Deer blood (five animal samples)c Tick A. americanum (five samples)c Tick A. americanumc THP-1 cell culture Canine platelets Vero cell culture Vero cell culture Tick Amblyomma ovale (five samples) Capybara spleen (five samples) Human blood (five samples) Water (three samples)
E. chaffeensis E. canis E. muris Ehrlichia sp. (HF565 strain) E. chaffeensis
No No No No No
14.3 12.7 18.5 19.1 31.0–37.4
E. chaffeensis
No
26.5–37.1
E. ewingii Anaplasma phagocytophilum Anaplasma platys Rickettsia bellii Rickettsia amblyommii Uninfected
No No No No No Yes
44.0 Neg. Neg. Neg. Neg. Mean: 17.0
Uninfected Uninfected Uninfected
Yes Yes Yes
Mean: 16.7 Mean: 16.2 Mean: 16.9
a b c
100–300 ng of DNA were used for each reaction. Each DNA sample (100–300 ng) was spiked with 50 ng of E. chaffeensis-infected DH82 cells DNA. DNA previously positive by the nested 16S rRNA PCR for Ehrlichia spp.
human, and of several Ehrlichia, Anaplasma and Rickettsia species (Table 2). 2.6. Confirming DNA amplification and DNA sequencing All real-time PCR products amplified from ticks or animals in the present study were confirmed by separating the PCR products using agarose (1.5%) gel electrophoresis. In addition, they were purified (ExoSAP-IT1 USB, Cleveland, USA) and the nucleotide sequence was determined by direct sequencing of the amplicon with both forward and reverse primers (Dsb-330 and Dsb-728) in an ABI automated sequencer (UTMB Protein Chemistry Core Facility, Galveston, TX). 3. Results 3.1. Brazilian clinical samples Four out of the five canine blood samples from Monte Negro County yielded expected real-time PCR products, which after sequencing (355-bp without primers) were 100% identical to each other and to the corresponding sequence of E. canis in GenBank (AF403710). In contrast, no Ehrlichia DNA was detected in the Brazilian samples of capybaras, humans, or ticks by the real-time PCR assay.
3.2. Validating the DNA extraction protocol To validate the DNA extraction protocol adopted in the present study, blood samples from three E. chaffeensis-infected deer, and one E. canis-infected dog, and one pool of E. canis-infected R. sanguineus ticks were subjected to DNA extraction with the same protocol used for the Brazilian clinical samples. All these samples showed positive results by the real-time PCR assay, which were confirmed by running the PCR products in 1.5% agarose gel electrophoresis. 3.3. Sensitivity and specificity of the real-time PCR assay The sensitivity of the real-time PCR assay was demonstrated to be a single copy of the E. chaffeensis dsb gene and 10 copies of the E. canis dsb gene as tested by serial dilutions of plasmids containing the entire dsb gene of E. chaffeensis or E. canis diluted in deer or tick DNA, respectively. The standard curve generated by the realtime PCR, running serial dilutions of E. chaffeensis dsb gene was represented by the equation: Y = 3.577X + 35.426, where Y is the Ct parameter (threshold cycle) and X is the log copy number of the DNA target. The standard curve generated by running serial dilutions of E. canis dsb gene was represented by the equation: Y = 3.609X + 44.721. The correlation coefficient for both equations was 0.999.
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The real-time PCR primers tested in the present study were able to amplify the dsb gene of all Ehrlicha spp. tested, which included E. chaffeensis, E. canis and E. muris from cell cultures, and Ehrlichia sp. (HF565 strain) from an experimentally infected mouse (Table 2). PCR products were not amplified from Rickettsia bellii, R. amblyommii, Anaplasma phagocytophilum DNA extracted from cell cultures, or A. platys DNA extracted from infected platelets. 3.4. Validating the real-time PCR assay All DNA samples from deer and ticks previously shown to contain ehrlichial DNA by the nested 16S rRNA PCR were confirmed as positive by this real-time PCR assay (Table 2). These included five individual deer samples naturally infected with E. chaffeensis, five individual A. americanum ticks naturally infected with E. chaffeensis, and one A. americanum naturally infected with E. ewingii. The E. ewingii-infected A. americanum tick yielded a 355-bp partial sequence of the dsb gene of E. ewingii, which has been deposited in GenBank (Accession number AY428950). The sequence similarities between the E. ewingii dsb partial sequence and the other five Ehrlichia species ranged from 76.6 to 78.0%. To test for the presence of possible PCR inhibitors, DNA samples of the Brazilian ticks, capybara spleen, and human blood (five individual samples of each) were spiked with E. chaffeensis DNA and tested by the realtime PCR assay. Three samples of water spiked with the same E. chaffeensis sample were used as control. All spiked samples were positive with similar Ct values, ranging from 16.2 to 17 (Table 2). Agarose gel electrophoresis of all the above samples that yielded positive results revealed a single band corresponding to the expected amplicon size (409 bp). 4. Discussion The present study reports for the first time E. canis infecting dogs in the Amazon region (Monte Negro County) of Brazil. Although CME is highly prevalent in the southern Brazilian states (Dagnone et al., 2003; Moreira et al., 2003), there has been no report in the northern states, which encompass the Brazilian Amazon region. The presence of E. canis in Monte Negro is certainly linked to the widespread occurrence of its vector, R. sanguineus, in the urban area of Monte Negro (Labruna et al., 2005). The dsb fragment sequenced from the Brazilian dogs in the present study was identical to the corresponding sequence of a North American isolate of E. canis
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(AF403710). As the dsb gene sequence identities between different Ehrlichia species varied from 74.6 to 91.8% (data not shown), the ehrlichial genotype detected in the four Brazilian dogs from the Amazon region refer, conclusively, to E. canis. Human ehrlichioses has emerged as an important disease after the discovery of E. chaffeensis and E. ewingii, both primarily associated with A. americanum ticks in the United States (Paddock and Childs, 2003). As this tick species is not established in South America, we tested representative species of the genus Amblyomma in Brazil. From the 47 Ixodidae tick species reported for Brazil, 33 (70%) belong to the genus Amblyomma (Labruna et al., 2005). For this reason, the present study tested only Amblyomma species, including the three species mostly associated with human infestations in Brazil (A. cajennense, A. ovale, and A. oblongoguttatum). It is noteworthy that the same clinical DNA samples from ticks tested in the present study were recently shown to contain detectable Rickettsia spp DNA by another real-time PCR assay (Labruna et al., 2004a,b), thus confirming the quality of our DNA samples. Furthermore, our sets of PCR assays have confirmed the suitability of our DNA extraction protocol and have allowed us to discard the presence of significant PCR inhibitors in the DNA test samples. Thus, as dog blood was the only clinical sample type that was shown to contain ehrlichial DNA in the present study (ticks, capybara, and human samples were all negative), it is possible that ehrlichiosis caused by E. chaffeensis or a closely related species is not a significant disease in the areas of the states of Rondoˆnia and Sa˜o Paulo evaluated in the present study. This result is reinforced by the fact that 75 blood samples collected from human febrile patients from Rondoˆnia, before any antibiotic therapy, showed no detectable ehrlichial DNA by the real-time PCR. In addition, our results also suggest that capybara, primary host for the ticks A. cajennense and A. dubitatum in Brazil, may not be commonly infected by Ehrlichia spp in Piracicaba County, state of Sa˜o Paulo. However, we are aware that we tested a limited number of samples and the surveillance of ehrlichiosis in Brazil must continue by testing more samples from different areas. In the present study, we used a highly sensitive quantitative real-time PCR that amplified a 409-bp fragment of the Ehrlichia spp. dsb gene, capable of detecting a single copy of E. chaffeensis or as few as 10 copies of E. canis. As the primers reported here amplify a specific 409-bp fragment, they can also be used in conventional PCR assays, allowing the diagnosis of the Ehrlichia species by sequencing the PCR product (as performed in the present study) or by restriction
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fragment length polymorphism (RFLP) patterns, yet to be evaluated. Furthermore, we decided to use this single step highly sensitive and specific real-time PCR because previous studies employing conventional 16S rRNA nested-PCR protocols have resulted in some unspecific amplification (16S rRNA fragments from other distantly related bacteria) (Lockhart et al., 1997; Whitlock et al., 2000), besides the high risk of sample crosscontamination and of detection of false positives inherent to any nested-PCR protocol (Kwok and Higuchi, 1989). No Ehrlichia species but E. canis has been reported in Brazil yet. Brazil is the fifth largest country of the world, with 26 states. As the present investigation evaluated samples from only two states, additional studies encompassing other geographic areas are needed to better elucidate the presence of ehrlichial agents in Brazil. Acknowledgments We thank R.E. Corstvet for providing A. platysinfected canine platelets, and J.S. Dumler for providing the A. phagocytophilum-infected cells. This work was supported by Fogarty International Center (grant D43TW00903 to DHWand MBL), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Brazil (grant 02/ 00644-0 to MBL), National Institutes of Allergy and Infectious Diseases (grant R01 AI044235-2 to WRD). References Allsopp, M.T.E.P., Allsopp, B.A., 2001. Novel Ehrlichia genotype detected in dogs in South Africa. J. Clin. Microbiol. 39, 4204– 4207. Anderson, B.E., Dawson, J.E., Jones, D.C., Wilson, K.H., 1991. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J. Clin. Microbiol. 29, 2838–2842. Calic, S.B., Galva˜o, M.A., Bacellar, F., Rocha, C.M., Mafra, C.L., Leite, R.C., Walker, D.H., 2004. Human ehrlichioses in Brazil: first suspect cases. Brazil J. Infect. Dis. 8, 259–262. Cao, W.C., Gao, Y.M., Zhang, P.H., Zhang, X.T., Dai, Q.H., Dumler, J.S., Fang, L.Q., Yang, H., 2000. Identification of Ehrlichia chaffeensis by nested PCR in ticks from southern China. J. Clin. Microbiol. 38, 2778–2780. Dagnone, A.S., Autran, H.S.M., Vidotto, M.C., Jojima, F.S., Vidotto, O., 2003. Ehrlichiosis in anemic, thrombocytopenic, or tickinfested dogs from a hospital population in south Brazil. Vet. Parasitol. 117, 285–290. Doyle, C.K., Labruna, M.B., Breitschwerdt, E.B., Tang, Y.W., Corstvet, R.E., Hegarty, B.C., Bloch, K.C., Li, P., Walker, D.H., McBride, J.W., 2005. Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. J. Mol. Diagn. 7, 504–510. Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of
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Short communication
A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil Marcelo B. Labruna a,b,*, Jere W. McBride a, Luis Marcelo A. Camargo c, Daniel M. Aguiar b, Michael J. Yabsley d, William R. Davidson d, Ellen Y. Stromdahl e, Phillip C. Williamson f, Roger W. Stich g, S. Wesley Long a, Erney P. Camargo c, David H. Walker a a
Department of Pathology, University of Texas Medical Branch, Galveston, TX 7755-0609, USA Departamento de Medicina Veterinaria Preventiva e Saude Animal, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil c Departamento de Parasitologia, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil d Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA e U.S. Army Center for Health Promotion and Preventive Medicine, APG, MD 21010, USA f DNA/Identity Laboratory, Department of Pathology and Anatomy, University of North Texas Health Science Center, Ft. Worth, TX 76107-2699, USA g Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210, USA b
Received 8 February 2006; received in revised form 1 July 2006; accepted 1 August 2006
Abstract A molecular epidemiologic investigation in two Brazilian states (Rondoˆnia and Sa˜o Paulo) was undertaken to determine if Ehrlichia species responsible for human and animal ehrlichioses in North America could be found in Brazilian vectors, potential natural mammalian reservoirs and febrile human patients with a tick bite history. Samples, including 376 ticks comprising 9 Amblyomma species, 29 capybara (Hydrochaeris hydrochaeris) spleens, 5 canine blood, and 75 human blood samples from febrile patients with history of tick bites were tested by a real-time PCR assay targeting a fragment of the Ehrlichia dsb gene. Ehrlichia DNA was not detected in any tick, capybara or human samples. In contrast, 4 out of 5 dogs contained Ehrlichia canis DNA in their blood, which were sequenced, representing the first report of E. canis infecting dogs in the Amazon region of Brazil. Further studies are needed to evaluate the presence of other agents of human and animal ehrlichioses in Brazil. # 2006 Elsevier B.V. All rights reserved. Keywords: Ehrlichia; Dsb gene; Real-time PCR; Ticks; Brazil
1. Introduction
* Corresponding author at: Departamento de Medicina Veterinaria Preventiva e Saude Animal, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitaria, Sa˜o Paulo, SP 05508-270, Brazil. Tel.: +55 11 3091 1394; fax: +55 11 3091 7928. E-mail address: [email protected] (M.B. Labruna). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.08.005
The genus Ehrlichia currently consists of five recognized species, which include E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium (Dumler et al., 2001). Other potential species, yet to be established in tissue culture, have been identified by molecular methods (Shibata et al., 2000; Wen et al., 2002; Inayoshi et al.,
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2004). All ehrlichial species infect vertebrate hosts and are transmitted by ticks. E. chaffeensis and E. ewingii are important agents of human disease in North America, whereas E. canis, E. ruminantium and E. ewingii are pathogens of major veterinary importance (Dumler et al., 2001). The discovery and emergence of E. chaffeensis as a human pathogen in the United States (Anderson et al., 1991; Paddock and Childs, 2003), and the improvements in molecular technologies during the last two decades has created an enormous interest in the global epidemiology and ecology of human ehrlichioses. Consequently, numerous studies investigating the ecological aspects of human ehrlichiosis in the United States have been reported (Paddock and Childs, 2003) and several new Ehrlichia genotypes found to infect ticks and animals have been reported outside the United States (Cao et al., 2000; Allsopp and Allsopp, 2001; Jenkins et al., 2001; Wen et al., 2002; Inayoshi et al., 2004). Currently, the only Ehrlichia species recognized in South America is E. canis, which is the primary etiologic agent of canine monocytic ehrlichiosis (CME) (Dumler et al., 2001; Moreira et al., 2003). In southern Brazil, CME is one of the most important infectious diseases of dogs (Dagnone et al., 2003; Moreira et al., 2003). Recent reports indicated that E. canis is a zoonotic agent causing asymptomatic or symptomatic infection in humans from Venezuela (Perez et al., 1996, 2005). In addition, there is serological evidence for human ehrlichiosis in Argentina and Brazil (Ripoli et al., 1999; Calic et al., 2004). Previously, no epidemiological study on ehrlichiosis (other than E. canis in urban areas) has been performed in Brazil. Thus, our study was a preliminary investigation, which tested the presence of ehrlichial DNA in tick, human, and animal samples from rural areas collected during other independent studies performed in Brazil (Labruna et al., 2004a,b, 2005). For this purpose, we used a highly sensitive and specific quantitative real-time PCR assay targeting the dsb gene of Ehrlichia species. 2. Materials and methods 2.1. Clinical samples from Brazil A total of 376 ticks were tested individually (n = 154) or in pools containing 2–10 ticks. The following tick species collected in the Amazon forest (Monte Negro and Gov. Jorge Teixeira Counties) in the state of Rondoˆnia (number of specimens in parenthesis) were tested: Amblyomma ovale (n = 121), Amblyomma cajennense (n = 41), Amblyomma scalpturatum (n = 35), Amblyomma naponense (n = 36), Amblyomma
oblongoguttatum (n = 30), Amblyomma coelebs (n = 10), and Amblyomma humerale (n = 6). The following tick species collected in the state of Sa˜o Paulo (number of specimens in parenthesis) were tested: Amblyomma dubitatum (=Amblyomma cooperi) (n = 32) and A. cajennense (n = 35) from Pedreira County; Amblyomma triste (n = 30) from Paulice´ia County. All ticks were collected as free-living in the vegetation except for A. humerale, which were collected from a tortoise during a previous study (Labruna et al., 2005). Capybara (Hydrocaheris hydrochaeris) spleens (n = 29) from Piracicaba County, state of Sa˜o Paulo, human blood samples (n = 75) from the rural area of Monte Negro County, state of Rondoˆnia, and canine blood samples (n = 5) from the urban area of Monte Negro were also tested. The human samples were collected from febrile patients with history of a tick exposure, prior to administration of antibiotic therapy. The canine samples were collected from domestic dogs showing infestation by Rhipicephalus sanguineus ticks and clinical signs compatible with CME (apathy, pale mucous membranes, and epistaxis). DNA from tick (whole tick body), capybara (10 mg of each spleen), canine, and human samples (100 ml of whole blood from each sample) was extracted using the DNeasy Tissue Kit (Qiagen, Chatsworth, CA) following the manufacturer’s protocols. 2.2. Testing Brazilian samples by real-time PCR assay All collected samples were tested by a real-time PCR assay as follows. Primers were designed based on the alignment of the 738-bp of the disulfide bond formation protein gene (dsb) of five Ehrlichia spp. available in GenBank (Table 1) using the CLUSTAL algorithm of the program MegAlign (DNAstar, Lasergene, Madison, WI). E. ewingii was the only species not included in the alignment because its dsb sequence was still undetermined. To design genus specific primers that could be used to amplify the dsb gene of all Ehrlichia species, we identified regions within the gene that were highly conserved among the different species. The nucleic acid sequence identities between the ehrlichial dsb genes varied from 74.6 to 91.8%, but two highly conserved regions were found in the gene alignment. A pair of primers designated as Dsb-330 and Dsb-728, were manually designed to correspond to these two conserved regions and amplify a 409-bp fragment of the Ehrlichia dsb (Table 1) (Doyle et al., 2005). To improve primer specificity (both primers have melting temperature >58 8C) and to minimize formation of primer dimers,
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Table 1 Nucleotide sequence alignment for the regions of the ehrlichial dsb gene corresponding to primers Dsb-330 and Dsb-728
the consensus primers were modified by arbitrary single nucleic acid substitutions (Dsb 330 position 1, A–G; Dsb 728 position 7, A–G) in the 50 region. Real-time PCR reactions were performed using a BioRad i-Cycler, with SYBR I green as the fluorescent dye. The amplification reaction, in a final volume of 50 ml, contained 25 ml of iQTMSYBR1Green I Supermix (Bio-Rad, Hercules, CA), 1 ml of each primer at 20 mM (final concentration of 400 nM), 18 ml of molecular grade water, and 5 ml of template containing 100–300 ng DNA. Primer concentrations were optimized in previous assays by spanning different initial concentrations of oligonucleotides. Real-time PCR cycling conditions were 1 cycle at 95 8C for 2 min followed by 50 cycles of 15 s at 95 8C, 30 s at 58 8C and 30 s at 72 8C, followed by an amplicon melt curve analysis. In each set of reactions, a positive control (E. chaffeensis DNA) and three negative water controls were included. 2.3. Testing the DNA extraction protocol To validate the DNA extraction protocol used for our Brazilian samples, blood samples from three E. chaffeensis-infected white-tail deer (provided by M.J.Y.), one blood sample from an experimentally E. canis-infected dog (provided by R.W.S.), and an experimentally E. canis-infected pool of two Rhipicephalus sanguineus ticks (provided by M.B.L.), all from the United States, were subjected to the same DNA extraction protocol cited above. These DNA samples were tested by the real-time PCR assay described above. 2.4. Testing the real-time PCR assay To validate the real-time PCR assay used for the Brazilian samples, the following DNA samples from
animals and ticks, previously shown by nested 16S rRNA PCR to contain ehrlichial DNA (Stromdahl et al., 2001; Yabsley et al., 2003; Long et al., 2004), were tested by the real-time PCR: DNA from peripheral blood of five white-tailed deer naturally infected with E. chaffeensis, one A. americanum tick naturally infected with E. ewingii, and five A. americanum ticks naturally infected with E. chaffeensis. 2.5. Testing the sensitivity and specificity of the real-time PCR assay To evaluate the sensitivity of the assay for detection of Ehrlichia spp. DNA, the entire dsb gene of E. chaffeensis (738 bp) was amplified as previously described (McBride et al., 2002) and cloned into a plasmid (pCR1T7 NT-TOPO1 TA Cloning Kit, Invitrogen, Carlsbad, CA). The DNA concentration of the purified plasmid was determined using a spectrophotometer at A260 (Perkin-Elmer MBA 2000, Norwalk, CT), and the number of plasmid copies (equivalent to the number of E. chaffeensis dsb gene copies) was calculated. Serial 10fold dilutions from 106 to 100 of plasmid containing the E. chaffeensis dsb gene were prepared in diluent containing DNA extracted from uninfected white-tailed deer blood. The plasmid dilutions mixed with 200 ng of deer DNA were used as standards in the real-time PCR assay and tested in duplicate. The same procedure described above was performed with the dsb gene of E. canis, except that uninfected R. sanguineus tick DNA was used as diluent, giving a final amount of 300 ng of tick DNA per standard template. Both DNA extractions of R. sanguineus ticks and deer blood were performed using the DNeasy Tissue Kit as cited above. The specificity of the real-time PCR assay was determined by testing genomic DNA of tick, capybara,
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Table 2 Samples tested to validate the real-time PCR assay DNA template sourcea
Infection status
Spiked with E. chaffeensis DNA b
Ct values
DH82 cell culture DH82 cell culture P388D1 cell culture Murine spleen Deer blood (five animal samples)c Tick A. americanum (five samples)c Tick A. americanumc THP-1 cell culture Canine platelets Vero cell culture Vero cell culture Tick Amblyomma ovale (five samples) Capybara spleen (five samples) Human blood (five samples) Water (three samples)
E. chaffeensis E. canis E. muris Ehrlichia sp. (HF565 strain) E. chaffeensis
No No No No No
14.3 12.7 18.5 19.1 31.0–37.4
E. chaffeensis
No
26.5–37.1
E. ewingii Anaplasma phagocytophilum Anaplasma platys Rickettsia bellii Rickettsia amblyommii Uninfected
No No No No No Yes
44.0 Neg. Neg. Neg. Neg. Mean: 17.0
Uninfected Uninfected Uninfected
Yes Yes Yes
Mean: 16.7 Mean: 16.2 Mean: 16.9
a b c
100–300 ng of DNA were used for each reaction. Each DNA sample (100–300 ng) was spiked with 50 ng of E. chaffeensis-infected DH82 cells DNA. DNA previously positive by the nested 16S rRNA PCR for Ehrlichia spp.
human, and of several Ehrlichia, Anaplasma and Rickettsia species (Table 2). 2.6. Confirming DNA amplification and DNA sequencing All real-time PCR products amplified from ticks or animals in the present study were confirmed by separating the PCR products using agarose (1.5%) gel electrophoresis. In addition, they were purified (ExoSAP-IT1 USB, Cleveland, USA) and the nucleotide sequence was determined by direct sequencing of the amplicon with both forward and reverse primers (Dsb-330 and Dsb-728) in an ABI automated sequencer (UTMB Protein Chemistry Core Facility, Galveston, TX). 3. Results 3.1. Brazilian clinical samples Four out of the five canine blood samples from Monte Negro County yielded expected real-time PCR products, which after sequencing (355-bp without primers) were 100% identical to each other and to the corresponding sequence of E. canis in GenBank (AF403710). In contrast, no Ehrlichia DNA was detected in the Brazilian samples of capybaras, humans, or ticks by the real-time PCR assay.
3.2. Validating the DNA extraction protocol To validate the DNA extraction protocol adopted in the present study, blood samples from three E. chaffeensis-infected deer, and one E. canis-infected dog, and one pool of E. canis-infected R. sanguineus ticks were subjected to DNA extraction with the same protocol used for the Brazilian clinical samples. All these samples showed positive results by the real-time PCR assay, which were confirmed by running the PCR products in 1.5% agarose gel electrophoresis. 3.3. Sensitivity and specificity of the real-time PCR assay The sensitivity of the real-time PCR assay was demonstrated to be a single copy of the E. chaffeensis dsb gene and 10 copies of the E. canis dsb gene as tested by serial dilutions of plasmids containing the entire dsb gene of E. chaffeensis or E. canis diluted in deer or tick DNA, respectively. The standard curve generated by the realtime PCR, running serial dilutions of E. chaffeensis dsb gene was represented by the equation: Y = 3.577X + 35.426, where Y is the Ct parameter (threshold cycle) and X is the log copy number of the DNA target. The standard curve generated by running serial dilutions of E. canis dsb gene was represented by the equation: Y = 3.609X + 44.721. The correlation coefficient for both equations was 0.999.
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The real-time PCR primers tested in the present study were able to amplify the dsb gene of all Ehrlicha spp. tested, which included E. chaffeensis, E. canis and E. muris from cell cultures, and Ehrlichia sp. (HF565 strain) from an experimentally infected mouse (Table 2). PCR products were not amplified from Rickettsia bellii, R. amblyommii, Anaplasma phagocytophilum DNA extracted from cell cultures, or A. platys DNA extracted from infected platelets. 3.4. Validating the real-time PCR assay All DNA samples from deer and ticks previously shown to contain ehrlichial DNA by the nested 16S rRNA PCR were confirmed as positive by this real-time PCR assay (Table 2). These included five individual deer samples naturally infected with E. chaffeensis, five individual A. americanum ticks naturally infected with E. chaffeensis, and one A. americanum naturally infected with E. ewingii. The E. ewingii-infected A. americanum tick yielded a 355-bp partial sequence of the dsb gene of E. ewingii, which has been deposited in GenBank (Accession number AY428950). The sequence similarities between the E. ewingii dsb partial sequence and the other five Ehrlichia species ranged from 76.6 to 78.0%. To test for the presence of possible PCR inhibitors, DNA samples of the Brazilian ticks, capybara spleen, and human blood (five individual samples of each) were spiked with E. chaffeensis DNA and tested by the realtime PCR assay. Three samples of water spiked with the same E. chaffeensis sample were used as control. All spiked samples were positive with similar Ct values, ranging from 16.2 to 17 (Table 2). Agarose gel electrophoresis of all the above samples that yielded positive results revealed a single band corresponding to the expected amplicon size (409 bp). 4. Discussion The present study reports for the first time E. canis infecting dogs in the Amazon region (Monte Negro County) of Brazil. Although CME is highly prevalent in the southern Brazilian states (Dagnone et al., 2003; Moreira et al., 2003), there has been no report in the northern states, which encompass the Brazilian Amazon region. The presence of E. canis in Monte Negro is certainly linked to the widespread occurrence of its vector, R. sanguineus, in the urban area of Monte Negro (Labruna et al., 2005). The dsb fragment sequenced from the Brazilian dogs in the present study was identical to the corresponding sequence of a North American isolate of E. canis
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(AF403710). As the dsb gene sequence identities between different Ehrlichia species varied from 74.6 to 91.8% (data not shown), the ehrlichial genotype detected in the four Brazilian dogs from the Amazon region refer, conclusively, to E. canis. Human ehrlichioses has emerged as an important disease after the discovery of E. chaffeensis and E. ewingii, both primarily associated with A. americanum ticks in the United States (Paddock and Childs, 2003). As this tick species is not established in South America, we tested representative species of the genus Amblyomma in Brazil. From the 47 Ixodidae tick species reported for Brazil, 33 (70%) belong to the genus Amblyomma (Labruna et al., 2005). For this reason, the present study tested only Amblyomma species, including the three species mostly associated with human infestations in Brazil (A. cajennense, A. ovale, and A. oblongoguttatum). It is noteworthy that the same clinical DNA samples from ticks tested in the present study were recently shown to contain detectable Rickettsia spp DNA by another real-time PCR assay (Labruna et al., 2004a,b), thus confirming the quality of our DNA samples. Furthermore, our sets of PCR assays have confirmed the suitability of our DNA extraction protocol and have allowed us to discard the presence of significant PCR inhibitors in the DNA test samples. Thus, as dog blood was the only clinical sample type that was shown to contain ehrlichial DNA in the present study (ticks, capybara, and human samples were all negative), it is possible that ehrlichiosis caused by E. chaffeensis or a closely related species is not a significant disease in the areas of the states of Rondoˆnia and Sa˜o Paulo evaluated in the present study. This result is reinforced by the fact that 75 blood samples collected from human febrile patients from Rondoˆnia, before any antibiotic therapy, showed no detectable ehrlichial DNA by the real-time PCR. In addition, our results also suggest that capybara, primary host for the ticks A. cajennense and A. dubitatum in Brazil, may not be commonly infected by Ehrlichia spp in Piracicaba County, state of Sa˜o Paulo. However, we are aware that we tested a limited number of samples and the surveillance of ehrlichiosis in Brazil must continue by testing more samples from different areas. In the present study, we used a highly sensitive quantitative real-time PCR that amplified a 409-bp fragment of the Ehrlichia spp. dsb gene, capable of detecting a single copy of E. chaffeensis or as few as 10 copies of E. canis. As the primers reported here amplify a specific 409-bp fragment, they can also be used in conventional PCR assays, allowing the diagnosis of the Ehrlichia species by sequencing the PCR product (as performed in the present study) or by restriction
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fragment length polymorphism (RFLP) patterns, yet to be evaluated. Furthermore, we decided to use this single step highly sensitive and specific real-time PCR because previous studies employing conventional 16S rRNA nested-PCR protocols have resulted in some unspecific amplification (16S rRNA fragments from other distantly related bacteria) (Lockhart et al., 1997; Whitlock et al., 2000), besides the high risk of sample crosscontamination and of detection of false positives inherent to any nested-PCR protocol (Kwok and Higuchi, 1989). No Ehrlichia species but E. canis has been reported in Brazil yet. Brazil is the fifth largest country of the world, with 26 states. As the present investigation evaluated samples from only two states, additional studies encompassing other geographic areas are needed to better elucidate the presence of ehrlichial agents in Brazil. Acknowledgments We thank R.E. Corstvet for providing A. platysinfected canine platelets, and J.S. Dumler for providing the A. phagocytophilum-infected cells. This work was supported by Fogarty International Center (grant D43TW00903 to DHWand MBL), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Brazil (grant 02/ 00644-0 to MBL), National Institutes of Allergy and Infectious Diseases (grant R01 AI044235-2 to WRD). References Allsopp, M.T.E.P., Allsopp, B.A., 2001. Novel Ehrlichia genotype detected in dogs in South Africa. J. Clin. Microbiol. 39, 4204– 4207. Anderson, B.E., Dawson, J.E., Jones, D.C., Wilson, K.H., 1991. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J. Clin. Microbiol. 29, 2838–2842. Calic, S.B., Galva˜o, M.A., Bacellar, F., Rocha, C.M., Mafra, C.L., Leite, R.C., Walker, D.H., 2004. Human ehrlichioses in Brazil: first suspect cases. Brazil J. Infect. Dis. 8, 259–262. Cao, W.C., Gao, Y.M., Zhang, P.H., Zhang, X.T., Dai, Q.H., Dumler, J.S., Fang, L.Q., Yang, H., 2000. Identification of Ehrlichia chaffeensis by nested PCR in ticks from southern China. J. Clin. Microbiol. 38, 2778–2780. Dagnone, A.S., Autran, H.S.M., Vidotto, M.C., Jojima, F.S., Vidotto, O., 2003. Ehrlichiosis in anemic, thrombocytopenic, or tickinfested dogs from a hospital population in south Brazil. Vet. Parasitol. 117, 285–290. Doyle, C.K., Labruna, M.B., Breitschwerdt, E.B., Tang, Y.W., Corstvet, R.E., Hegarty, B.C., Bloch, K.C., Li, P., Walker, D.H., McBride, J.W., 2005. Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. J. Mol. Diagn. 7, 504–510. Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of
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