- Email: [email protected]
Detection of Ehrlichia chaffeensis in Brazilian marsh deer (Blastocerus dichotomus)
Veterinary Parasitology 139 (2006) 262–266 www.elsevier.com/locate/vetpar
Short communication
Detection of Ehrlichia chaffeensis in Brazilian marsh deer (Blastocerus dichotomus) Rosangela Zacarias Machado a,*, Jose´ Maurı´cio B. Duarte a, Ana Silvia Dagnone a, Matias Pablo J. Szabo´ b a
Universidade Estadual Paulista, UNESP-Campus de Jaboticabal, Departamento de Patologia Veterina´ria, FCAV/UNESP-Rodovia de Acesso Paulo Donato Castellane, s/n – Jaboticabal, SP, Brazil b Universidade Federal de Uberlaˆndia, UFU-Uberlaˆndia, MG, Brazil Received 14 April 2005; received in revised form 26 January 2006; accepted 24 February 2006
Abstract Ehrlichia chaffeensis was detected for the first time in blood samples from Brazilian marsh deers (Blastocerus dichotomus) captured in the marshes of Parana River in Southeast Brazil in 1998. Seven EDTA-blood samples from deers were analyzed by PCR and nested PCR for presence of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Neoriickettsia risticii, Anaplasma phagocytophilum and Anaplasma marginale. Three samples showed positive reactions for E. chaffeensis and Anaplasma marginale. None contained detectable A. phagocytophilum, E. ewingii, E. canis or Neorickettsia risticii DNA. In Brazil, the wild marsh deer may be a natural reservoir of the agents that cause human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis. # 2006 Elsevier B.V. All rights reserved. Keywords: Ehrlichia chaffeensis; Anaplasma marginale; Marsh Deer; PCR; nPCR; Brazil
1. Introduction Family Anaplasmataceae (formerly Ehrlichieae) comprise a group of rickettsial agents that are obligate intracellular bacteria and reside within a cytoplasmic vacuole of infected eukaryotic cells (Dumler et al., 2001). * Corresponding author. Tel.: +55 16 32092662; fax: +55 16 3202 2978. E-mail address: [email protected] (R.Z. Machado).
Anaplasma marginale and A. phagocytophilum are closely related, but only ruminants are cited as susceptible hosts for A. marginale (Kutler, 1994). In contrast, A. phagocytophilum has zoonotic potential and infects ruminants, dogs, horses, small rodents and humans (Dumler et al., 2001). At least three known agents in the United States of America, E. chaffeensis, E. ewingii and A. phagocytophilum cause human disease (Dawson et al., 1991; Dumler et al., 2001). The presence of E. chaffeensis agent in some wild animals and Ixodid ticks in North America were demonstraded
0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.02.038
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
263
also described in humans (Buller et al., 1999). Whitetailed deers (Odocoileus virginianus) are believed to be natural reservoirs of E. chaffeensis (Lockhart et al., 1997); however, deer can be infected by several species within the genera Ehrlichia and Anaplasma that result in potential cross-reactions among otherwise specific serologic tests (Unver et al., 1999). Immunoserologic evidence of exposure to E. chaffeensis, E. canis and A. phagocytophilum has been
(Lockhart et al., 1997; Anderson et al., 1993) and the likelihood of the ticks could act as reservoirs still remained to be evaluated. Human ehrlichiosis was first reported in 1987 and since then the causative agents of monocytotropic ehrlichiosis, E. chaffeensis, and of granulocytotropic anaplasmosis, A. phagocytophilum, have been described in many parts of United States (Walker and Dumler, 1996). E. ewingii, another granulocytotropic agent of ehrlichiosis in dogs was
Table 1 Oligonucleotide sequences of Anaplasmataceae agents, amplicon size and reference of each protocol Anaplasmataceae agents
Sequence of primers
Ehrlichia spp ECC ECB
50 - aga acg aac gct ggc ggc aag cc - 30 50 - cgt att acc gcg gct gct ggc - 30
478
E. canis Can GA1UR
50 - caa tta ttt ata gcc tct ggc tat agg a - 30 ) 50 - gac ttt gcc ggg act tct tct - 30
358
E. chaffeensis Chaff GA1UR
50 - caa ttg ctt ata acc ttt tgg tta taa at - 30 50 - gac ttt gcc ggg act tct tct - 30
410
A. phagocytophilum gE3a gE10R gE2 gE9F
50 50 50 50
932
– – – –
Amplicon size (bp)
cac atg caa gtc gaa cgg att att c - 30 ttc cgt taa gaa gga tct aat ctc c - 30 ggc agt att aaa agc agc tcc agg - 30 aac gga tta ttc ttt ata gct tgc t - 30
A. phagocytophilum (msp2-gene) MSP 465f 50 - tga tgt tgt tac tgg aca ga - 30 MSP 980r 50 - cac cta acc ttc ata aga a - 30
A. marginale (msp5) Ext F Ext R Int F
50 – gca tag cct ccg cgt ctt tc - 30 50 – tcc tcg cct tgc ccc tca ga -30 50 – tac acg tgc cta ccg act ta - 30
Neorickettsia risticii ER3-F ER2-R ER 3a-F ER2a-R
5’-atttgagagtttgatcctgg-3’ 5’-gttttaaatgcagttcttgg-3’ 5’-ctagcggtaggcttaac-3’ 5’-cacacctaacttacggg-3’
E. ewingii 8F 1448R
50 - agt ttg atc atg gct cag-30 50 - cca tgg cgt gac ggg cag tgt g-30
ewingii GA1UR
0
Persing (1996) Kocan et al. (2000)
Massung et al. (1998)
546
550
A. phagocytophilum (16S rRNA gene) 8F 50 - agt ttg atc atg gct cag - 30 1448R 50 - cca tgg cgt gac ggg cag tgt g - 30 equi 50 - tta ttc ttt ata gct tgc tat aaa g -30 GA1UR 50 - gac ttt gcc ggg act tct tct - 30
Reference
1440
Caspersen et al. (2002)
Persing (1996)
406
458
Torioni et al. (1998)
345
599
Chae et al. (2003)
529 1440 Persing (1996) 0
5 - caa ttc cta aat agt ctc tga cta tt-3 50 - gac ttt gcc ggg act tct tct-30
412
264
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
reported in Venezuela and Brazil (Arraga-Alvarado et al., 1996; Galva˜o et al., 2002; Calici et al., 2004). Recently, Szabo´ et al. (2003) found the tick species Amblyomma cajennense, A. triste, Anocentor nitens and Boophilus microplus on wild marsh deer (B. dichotomus) captured before and after closure of the Porto – Primavera Hydroelectric Power Station at the border of Sa˜o Paulo and Mato Grosso do Sul states. Because there is no evidence of anaplasmal agents in wild marsh deer from this area, the main purpose of this study was to examine for the presence of E. chaffeensis, E. canis, E. ewingii, Neorickettsia risticii, A. phagocytophilum and A. marginale in blood from these animals using molecular methods.
2. Materials and methods In Brazil, Porto Primavera Hydroelectric Power Station is located between the southwest of Sa˜o Paulo State and east of Mato Grosso do Sul State. Marsh deer were captured as described by Duarte et al. (2001) and biological samples (blood, feces, semen, fur and skin) were simultaneously obtained for different studies as previously described by Szabo´ et al. (2003). DNA was extracted from 200 mL of whole blood of 7 animals using the QIAamp DNA Blood Mini Kit (QIAGEN TM ), according to the manufacturer’s instructions. Each extracted DNA was used as template in 50 mL reaction mixtures containing 10X PCR buffer, 1.5 mM MgCl2, 10 mM deoxynucleotide triphosphate (dNTP) mixture, and DNA Taq polymerase (InvitrogenTM), with separate primers for E. chaffeensis, E. canis,
E. ewingii, A. phagocytophilum, A. marginale or N. risticii (Table 1) for the first and nested amplifications (nPCR). The PCR and nPCR amplifications were performed in a Gradient cycler (Perkin-ElmerTM model PT-200). In each set of amplifications, both positive and negative controls were included. Sequences confirmation were performed and accession numbers were listed at the end of the results in this manuscript.
3. Results and discussion Three of the seven (42.86%) marsh deer were naturally infected with E. chaffeensis, providing the first evidence infection of deer in Brazil (Fig. 1). A. marginale genomic material was also amplified in two of the three E. chaffeensis-positive samples and one was only positive for the former agent. No deer were infected with E. canis, E. ewingii, N. risticii or A. phagocytophilum on the basis of PCR and nPCR. Sequence analysis of our E. chaffeensis positive samples (accession number DQ345720) showed higher than 97% similarity with some sequences deposited in GenBank (AF305074; AY350424). The tick B. microplus is a potential vector of this agent and it infests both cattle and deer in this area (Duarte et al., 2001). These data also confirm previous investigations that suggested natural deer infection after the detection of antibodies to A. marginale in some areas in Brazil (Duarte et al., 2001). E. chaffeensis causes human monocytotropic ehrlichiosis, a tick-borne zoonosis in the southern,
Fig. 1. Agarose gel electrophoresis demonstration of Ehrlichia chaffeensis PCR (A) and nested PCR (B) products obtained after amplification with primer pair ECC/ECB and GAIUR/Chaf. A. Lane M: molecular standard size; Lane 1, positive control (458 bp); Lane 2 through 7 Deer blood samples and Lane 8, negative control. B. Lane M: molecular standard size; Lane 9, positive control (410 bp); Lanes 10, 14 and 15, negative Deer blood samples; Lanes 11–13 positive Deer blood samples and Lane 16, negative control.
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
south-central and mid-Atlantic regions of the United States (Eng et al., 1990; Dawson et al., 1991). The main reservoir is the white-tailed deer (Odocoileus virginianus) (Ewing et al., 1995; Lockhart et al., 1997), the most important host for adult and immature stages of Amblyomma americanum (Paddock and Childs, 2003). Moreover, E. chaffeensis DNA was found in 71% of coyotes among 21 animals examined, suggesting that this bacterium commonly infects free ranging coyotes in Oklahoma, USA (Kocan et al., 2000). However, goats, dogs, red foxes, raccoons, opossums and rodents can also serve as hosts for all stages of Amblyomma americanum (Paddock and Childs, 2003), and that would suggest they may be involved in the natural maintenance of E. chaffeensis (Williams et al., 2002). The wild marsh-deer from Southeast Brazil hosts four species of ticks: A. cajennense, A. triste, Anocentor nitens and Boophilus microplus (Szabo´ et al., 2003), but little is known about the vector–pathogen relationships among these ticks. The demonstration of Anaplasma marginale by serological tests and direct detection indicate a role for Brazilian deer and B. microplus as reservoirs and candidate- vectors, respectively (Duarte et al., 2001; Sachi et al., 2004). The two co-infected deers of this study were heavily infested by B. microplus and belong to the same geographical area. Outside of the U.S., human ehrlichial infections were reported in Mexico and Argentina based on serological data using E. chaffeensis as antigen (Gongora-Biachi et al., 1999; Ripoll et al., 1999). Isolation and antigenic and genetic characterization of E. canis isolated from a human in Venezuela was reported, although the patient lacked any clinical manifestations (Unver et al., 2001). Furthermore, in 2002, antibodies to E. chaffeensis were detected for the first time in a serologic survey of dogs from a spotted fever-endemic area in Minas Gerais State, Brazil (Galva˜o et al., 2002), where suspected cases of human monocytotropic ehrlichiosis were recently serologically detected (Calici et al., 2004). Since E. chaffeensis can potentially cross-react with E. canis, A. phagocytophilum and E. ewingii (Buller et al., 1999; Unver et al., 1999), it is important to determine specifically which Anaplasmataceae is present and potentially able to infect humans in Brazil. According to Szabo´ et al. (2003), the reservoir formed by the
265
Hydroelectric Power Station reduced the natural habitat of many species, especially those living in marsh areas, thereby affecting wildlife in a multitude of ways. It is noteworthy that changes in the host– vector ecology could expose human populations to ehrlichial diseases as human recreational and occupational activities facilitate increased contact with wild animals or when natural ecological cycles are disturbed. In conclusion, this study provides the first documentation of E. chaffeensis and A. marginale presence in marsh deer in Brazil and suggests a role for these animals as potential reservoirs, increasing risk for human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis. Also, the low number of deer blood samples could be explained by a random choice of a higher number group of animals which are near three hundred. However, we are working on the whole group at this time and sequencing of all positive samples will be performed. Acknowledgments J. Stephen Dumler for the Ehrlichia chaffeensis, Anaplasma phagocytophilum and N. risticii positive controls. Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for the finantial support. References Anderson, B.E., Sims, K.G., Olson, J.G., Childs, J.E., Piesman, J.F., Happ, C.M., Maupin, G.O., Johson, J.B., 1993. Amblyomma americanum: a potential vector of human ehrlichiosis. Am. J. Med. Hyg. 49, 239–244. Arraga-Alvarado, C., Montero-Ojeda, M., Bernardoni, A., Anderson, B.E., Parra, O., 1996. Human ehrlichiosis: report of the first case in Venezuela. Invest. Clin. 37, 35–49. Buller, R.S., Arens, M., Hmiel, S.P., Paddock, C.D., Sumner, J.W., Rikihisa, Y., Unver, Galdreault, K.E.E.N.E.R., Manian, F.A., Liddell, A.M., Schmulewitz, N., Storch, G.A., 1999. Ehrlichia ewingii recognized agent of human ehrlichiosis. N. Engl. J. Med. 341, 148–155. Calici, S.B., Galva˜o, M.A.M., Bacellar, F., Rocha, C.M.B.M., Mafra, C.L., Leite, R.C., Walker, D.H., 2004. Human ehrlichiosis in Brazil: first suspect cases. Braz. J. Infect. Dis. 8, 259– 262. Caspersen, K., Park, J.-H., Patil, S., Dumler, J.S., 2002. Genetic variability and stability of Anaplasma phagocytophila msp2 (p44). Infect. Immun. 70, 1230–1234.
266
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
Chae, J.S., Kim, M., Kim, M., Cho, Y., Park, B., 2003. Prevalence and sequence analyses of Neorickettsia risticii. Ann. NY. Acad. Sci. 990, 248–256. Dawson, J.E., Anderson, B.E., Fisbein, D.B., Sanchez, J.L., Goldsmith, C.S., Wilson, K.H., Duntley, C.W., 1991. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human Ehrlichiosis. J. Clin. Microbiol. 29, 2741–2745. Duarte, J.M.B., Merino, M.L., Gozales, M.S., Nunes, A.L.V., Garcia, J.M., Szabo´, M.J.P., Pandolfi, J.R., Arantes, I.G., Nascimento, A.A., Machado, R.Z.M., Araujo, J.P., Cata˜o-Dias, J.L., Werther, K., Garcia, J.E., Girio, J.S., Matushima, E.R., 2001. Order Artiodactyla family cervidae (Deer). Biology and medicine. In: Fowler, M.E., Cubas, Z.S. (Eds.), Biology, Medicine and Surgery of South American wild animals. Iowa State University Press, Ames, IA, pp. 402–422. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51, 2145–2165. Eng, T.R., Fishbein, D.B., Dawson, J.E., Greene, C.R., Redus, M., 1990. Survailence of human erhlichiosis in the United States. Ann. NY. Acad. Sci. 590, 306–307. Ewing, S.A., Dawson, J.E., Kocan, A.A., Barker, R.W., Warner, C.K., Panciera, R.J., Fox, J.C., Kocan, K.M., Blouin, E.F., 1995. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 32, 368–374. Galva˜o, M.A.M., Lamounier, J.A., Bonono, E., Tropia, M.S., Rezende, E.G., Calic, S.B., Chamone, C.B., Machado, M.C., Otoni, M.E.A., Leite, R.C., Caram, C., Mafra, C.L., Walker, D.H., 2002. Emerging and reemerging rickettsiosis in an endemic area of Minas Gerais State, Brazil. Cadernos de Sau´de Pu´blica 18, 1593–1597. Gongora-Biachi, R.A., Zavala-Velazquez, J., Castro-Sansores, C.J., Gonzalez-Martinez, P., 1999. First case of human ehrlichiosis in Mexico. E. I. D. 5, 481. Kocan, A., Levesque, G.C., Whitworth, L.C., Murphy, G.L., Ewing, S.A., Barker, R.W., 2000. Naturally occuring Ehrlichia chaffeensis infection in coyotes from Oklahoma. E. I. D. 6, 477–480. Kutler, K.L., 1994. Anaplasma infections in wild and domestic ruminants: a review. J. Wildl. Dis. 20, 12–20.
Lockhart, J.M., Davidson, D.E., Stallknecht, J.E., Dawson, J.E., Howerth, E.W., 1997. Isolation of Ehrlichia chaffeensis from wild white-tailed deer (Odocoileus virginianus) confirms their role as natural reservoir hosts. J. Clin. Microbiol. 35, 1681–1686. Massung, R., Slater, K., Owens, J.H., Nicholson, W.L., Mather, T.N., Solberg, V.B., Olson, J.G., 1998. Nested PCR assay for detection of Granulocytic Ehrlichiae. J. Clin. Microbiol. 36, 1090–1095. Paddock, C.D., Childs, J.E., 2003. Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin. Microbiol. Rev. 16, 37–64. Persing, D.H., 1996. PCR protocols for emerging infectious diseases. In: A supplement to Diagnostic Molecular Microbiology: Principles and Applications, ASM Press, Washington, DC. Ripoll, C.M., Remondegui, C.E., Ordonez, G., Arazamende, R., Fusaro, H., Ryman, M.J., Paddock, C.D., Zaki, S.R., Olson, J.G., Santos-Buch, C.A., 1999. Evidence of Rickettsial spotted fever and ehrlichial infections in a subtropical territory of Jujuy. Argentina Am. J. Trop. Med. Hyg. 61, 350–354. Szabo´, M.P.J., Labruna, M.B., Pereira, M.C., Duarte, J.M.B., 2003. Ticks (Acari:Ixodidade) in Wild Marsh Deer (Blastocerus dichotomus) from Southeast Brazil: Infestation before and after habitat loss. J. Med. Entomol. 40, 268–274. Torioni, E.S., Knowles, D.P., McGuire, T.C., Palmer, G.H., Suarez, C.E., Mcelwain, T.F., 1998. Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5. J. Clin. Microbiol. 36, 777–782. Unver, A., Rikihisa, Y., Ohashi, N., Cullman, L.C., Buller, R., Storch, G.A., 1999. Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and –negative human sera by using native and recombinant E. chaffeensis and E. canis antigens. J. Clin. Microbiol. 37, 3888– 3895. Unver, A., Felek, S., Paddock, N., Zhi, N., Horowitz, H.W., Wormser, G.P., Cullman, L.C., Rikihisa, Y., 2001. Western blot analysis of sera reactive to human monocytic ehrlichiosis and human granulocytic ehrlichiosis agents. J. Clin. Microbiol. 39, 3982–3986. Walker, D.H., Dumler, J.S., 1996. Emergence of the Ehrlichioses as Human Health Problems. E. I. D. 2, 18–29. Williams, C.V., Steenhouse, J.L.V., Bradley, J.M., Hancock, S.I., Hegarty, B.C., Breitschwerdt, E.B., 2002. Naturally occuring Ehrlichia chaffeensis infection in two prosimian primate species: Ring-tailed Lemurs (Lemur catta) and Ruffed Lemurs (Varecia variegata). E. I. D. 8, 1497–1500.
Short communication
Detection of Ehrlichia chaffeensis in Brazilian marsh deer (Blastocerus dichotomus) Rosangela Zacarias Machado a,*, Jose´ Maurı´cio B. Duarte a, Ana Silvia Dagnone a, Matias Pablo J. Szabo´ b a
Universidade Estadual Paulista, UNESP-Campus de Jaboticabal, Departamento de Patologia Veterina´ria, FCAV/UNESP-Rodovia de Acesso Paulo Donato Castellane, s/n – Jaboticabal, SP, Brazil b Universidade Federal de Uberlaˆndia, UFU-Uberlaˆndia, MG, Brazil Received 14 April 2005; received in revised form 26 January 2006; accepted 24 February 2006
Abstract Ehrlichia chaffeensis was detected for the first time in blood samples from Brazilian marsh deers (Blastocerus dichotomus) captured in the marshes of Parana River in Southeast Brazil in 1998. Seven EDTA-blood samples from deers were analyzed by PCR and nested PCR for presence of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Neoriickettsia risticii, Anaplasma phagocytophilum and Anaplasma marginale. Three samples showed positive reactions for E. chaffeensis and Anaplasma marginale. None contained detectable A. phagocytophilum, E. ewingii, E. canis or Neorickettsia risticii DNA. In Brazil, the wild marsh deer may be a natural reservoir of the agents that cause human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis. # 2006 Elsevier B.V. All rights reserved. Keywords: Ehrlichia chaffeensis; Anaplasma marginale; Marsh Deer; PCR; nPCR; Brazil
1. Introduction Family Anaplasmataceae (formerly Ehrlichieae) comprise a group of rickettsial agents that are obligate intracellular bacteria and reside within a cytoplasmic vacuole of infected eukaryotic cells (Dumler et al., 2001). * Corresponding author. Tel.: +55 16 32092662; fax: +55 16 3202 2978. E-mail address: [email protected] (R.Z. Machado).
Anaplasma marginale and A. phagocytophilum are closely related, but only ruminants are cited as susceptible hosts for A. marginale (Kutler, 1994). In contrast, A. phagocytophilum has zoonotic potential and infects ruminants, dogs, horses, small rodents and humans (Dumler et al., 2001). At least three known agents in the United States of America, E. chaffeensis, E. ewingii and A. phagocytophilum cause human disease (Dawson et al., 1991; Dumler et al., 2001). The presence of E. chaffeensis agent in some wild animals and Ixodid ticks in North America were demonstraded
0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.02.038
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
263
also described in humans (Buller et al., 1999). Whitetailed deers (Odocoileus virginianus) are believed to be natural reservoirs of E. chaffeensis (Lockhart et al., 1997); however, deer can be infected by several species within the genera Ehrlichia and Anaplasma that result in potential cross-reactions among otherwise specific serologic tests (Unver et al., 1999). Immunoserologic evidence of exposure to E. chaffeensis, E. canis and A. phagocytophilum has been
(Lockhart et al., 1997; Anderson et al., 1993) and the likelihood of the ticks could act as reservoirs still remained to be evaluated. Human ehrlichiosis was first reported in 1987 and since then the causative agents of monocytotropic ehrlichiosis, E. chaffeensis, and of granulocytotropic anaplasmosis, A. phagocytophilum, have been described in many parts of United States (Walker and Dumler, 1996). E. ewingii, another granulocytotropic agent of ehrlichiosis in dogs was
Table 1 Oligonucleotide sequences of Anaplasmataceae agents, amplicon size and reference of each protocol Anaplasmataceae agents
Sequence of primers
Ehrlichia spp ECC ECB
50 - aga acg aac gct ggc ggc aag cc - 30 50 - cgt att acc gcg gct gct ggc - 30
478
E. canis Can GA1UR
50 - caa tta ttt ata gcc tct ggc tat agg a - 30 ) 50 - gac ttt gcc ggg act tct tct - 30
358
E. chaffeensis Chaff GA1UR
50 - caa ttg ctt ata acc ttt tgg tta taa at - 30 50 - gac ttt gcc ggg act tct tct - 30
410
A. phagocytophilum gE3a gE10R gE2 gE9F
50 50 50 50
932
– – – –
Amplicon size (bp)
cac atg caa gtc gaa cgg att att c - 30 ttc cgt taa gaa gga tct aat ctc c - 30 ggc agt att aaa agc agc tcc agg - 30 aac gga tta ttc ttt ata gct tgc t - 30
A. phagocytophilum (msp2-gene) MSP 465f 50 - tga tgt tgt tac tgg aca ga - 30 MSP 980r 50 - cac cta acc ttc ata aga a - 30
A. marginale (msp5) Ext F Ext R Int F
50 – gca tag cct ccg cgt ctt tc - 30 50 – tcc tcg cct tgc ccc tca ga -30 50 – tac acg tgc cta ccg act ta - 30
Neorickettsia risticii ER3-F ER2-R ER 3a-F ER2a-R
5’-atttgagagtttgatcctgg-3’ 5’-gttttaaatgcagttcttgg-3’ 5’-ctagcggtaggcttaac-3’ 5’-cacacctaacttacggg-3’
E. ewingii 8F 1448R
50 - agt ttg atc atg gct cag-30 50 - cca tgg cgt gac ggg cag tgt g-30
ewingii GA1UR
0
Persing (1996) Kocan et al. (2000)
Massung et al. (1998)
546
550
A. phagocytophilum (16S rRNA gene) 8F 50 - agt ttg atc atg gct cag - 30 1448R 50 - cca tgg cgt gac ggg cag tgt g - 30 equi 50 - tta ttc ttt ata gct tgc tat aaa g -30 GA1UR 50 - gac ttt gcc ggg act tct tct - 30
Reference
1440
Caspersen et al. (2002)
Persing (1996)
406
458
Torioni et al. (1998)
345
599
Chae et al. (2003)
529 1440 Persing (1996) 0
5 - caa ttc cta aat agt ctc tga cta tt-3 50 - gac ttt gcc ggg act tct tct-30
412
264
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
reported in Venezuela and Brazil (Arraga-Alvarado et al., 1996; Galva˜o et al., 2002; Calici et al., 2004). Recently, Szabo´ et al. (2003) found the tick species Amblyomma cajennense, A. triste, Anocentor nitens and Boophilus microplus on wild marsh deer (B. dichotomus) captured before and after closure of the Porto – Primavera Hydroelectric Power Station at the border of Sa˜o Paulo and Mato Grosso do Sul states. Because there is no evidence of anaplasmal agents in wild marsh deer from this area, the main purpose of this study was to examine for the presence of E. chaffeensis, E. canis, E. ewingii, Neorickettsia risticii, A. phagocytophilum and A. marginale in blood from these animals using molecular methods.
2. Materials and methods In Brazil, Porto Primavera Hydroelectric Power Station is located between the southwest of Sa˜o Paulo State and east of Mato Grosso do Sul State. Marsh deer were captured as described by Duarte et al. (2001) and biological samples (blood, feces, semen, fur and skin) were simultaneously obtained for different studies as previously described by Szabo´ et al. (2003). DNA was extracted from 200 mL of whole blood of 7 animals using the QIAamp DNA Blood Mini Kit (QIAGEN TM ), according to the manufacturer’s instructions. Each extracted DNA was used as template in 50 mL reaction mixtures containing 10X PCR buffer, 1.5 mM MgCl2, 10 mM deoxynucleotide triphosphate (dNTP) mixture, and DNA Taq polymerase (InvitrogenTM), with separate primers for E. chaffeensis, E. canis,
E. ewingii, A. phagocytophilum, A. marginale or N. risticii (Table 1) for the first and nested amplifications (nPCR). The PCR and nPCR amplifications were performed in a Gradient cycler (Perkin-ElmerTM model PT-200). In each set of amplifications, both positive and negative controls were included. Sequences confirmation were performed and accession numbers were listed at the end of the results in this manuscript.
3. Results and discussion Three of the seven (42.86%) marsh deer were naturally infected with E. chaffeensis, providing the first evidence infection of deer in Brazil (Fig. 1). A. marginale genomic material was also amplified in two of the three E. chaffeensis-positive samples and one was only positive for the former agent. No deer were infected with E. canis, E. ewingii, N. risticii or A. phagocytophilum on the basis of PCR and nPCR. Sequence analysis of our E. chaffeensis positive samples (accession number DQ345720) showed higher than 97% similarity with some sequences deposited in GenBank (AF305074; AY350424). The tick B. microplus is a potential vector of this agent and it infests both cattle and deer in this area (Duarte et al., 2001). These data also confirm previous investigations that suggested natural deer infection after the detection of antibodies to A. marginale in some areas in Brazil (Duarte et al., 2001). E. chaffeensis causes human monocytotropic ehrlichiosis, a tick-borne zoonosis in the southern,
Fig. 1. Agarose gel electrophoresis demonstration of Ehrlichia chaffeensis PCR (A) and nested PCR (B) products obtained after amplification with primer pair ECC/ECB and GAIUR/Chaf. A. Lane M: molecular standard size; Lane 1, positive control (458 bp); Lane 2 through 7 Deer blood samples and Lane 8, negative control. B. Lane M: molecular standard size; Lane 9, positive control (410 bp); Lanes 10, 14 and 15, negative Deer blood samples; Lanes 11–13 positive Deer blood samples and Lane 16, negative control.
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
south-central and mid-Atlantic regions of the United States (Eng et al., 1990; Dawson et al., 1991). The main reservoir is the white-tailed deer (Odocoileus virginianus) (Ewing et al., 1995; Lockhart et al., 1997), the most important host for adult and immature stages of Amblyomma americanum (Paddock and Childs, 2003). Moreover, E. chaffeensis DNA was found in 71% of coyotes among 21 animals examined, suggesting that this bacterium commonly infects free ranging coyotes in Oklahoma, USA (Kocan et al., 2000). However, goats, dogs, red foxes, raccoons, opossums and rodents can also serve as hosts for all stages of Amblyomma americanum (Paddock and Childs, 2003), and that would suggest they may be involved in the natural maintenance of E. chaffeensis (Williams et al., 2002). The wild marsh-deer from Southeast Brazil hosts four species of ticks: A. cajennense, A. triste, Anocentor nitens and Boophilus microplus (Szabo´ et al., 2003), but little is known about the vector–pathogen relationships among these ticks. The demonstration of Anaplasma marginale by serological tests and direct detection indicate a role for Brazilian deer and B. microplus as reservoirs and candidate- vectors, respectively (Duarte et al., 2001; Sachi et al., 2004). The two co-infected deers of this study were heavily infested by B. microplus and belong to the same geographical area. Outside of the U.S., human ehrlichial infections were reported in Mexico and Argentina based on serological data using E. chaffeensis as antigen (Gongora-Biachi et al., 1999; Ripoll et al., 1999). Isolation and antigenic and genetic characterization of E. canis isolated from a human in Venezuela was reported, although the patient lacked any clinical manifestations (Unver et al., 2001). Furthermore, in 2002, antibodies to E. chaffeensis were detected for the first time in a serologic survey of dogs from a spotted fever-endemic area in Minas Gerais State, Brazil (Galva˜o et al., 2002), where suspected cases of human monocytotropic ehrlichiosis were recently serologically detected (Calici et al., 2004). Since E. chaffeensis can potentially cross-react with E. canis, A. phagocytophilum and E. ewingii (Buller et al., 1999; Unver et al., 1999), it is important to determine specifically which Anaplasmataceae is present and potentially able to infect humans in Brazil. According to Szabo´ et al. (2003), the reservoir formed by the
265
Hydroelectric Power Station reduced the natural habitat of many species, especially those living in marsh areas, thereby affecting wildlife in a multitude of ways. It is noteworthy that changes in the host– vector ecology could expose human populations to ehrlichial diseases as human recreational and occupational activities facilitate increased contact with wild animals or when natural ecological cycles are disturbed. In conclusion, this study provides the first documentation of E. chaffeensis and A. marginale presence in marsh deer in Brazil and suggests a role for these animals as potential reservoirs, increasing risk for human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis. Also, the low number of deer blood samples could be explained by a random choice of a higher number group of animals which are near three hundred. However, we are working on the whole group at this time and sequencing of all positive samples will be performed. Acknowledgments J. Stephen Dumler for the Ehrlichia chaffeensis, Anaplasma phagocytophilum and N. risticii positive controls. Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for the finantial support. References Anderson, B.E., Sims, K.G., Olson, J.G., Childs, J.E., Piesman, J.F., Happ, C.M., Maupin, G.O., Johson, J.B., 1993. Amblyomma americanum: a potential vector of human ehrlichiosis. Am. J. Med. Hyg. 49, 239–244. Arraga-Alvarado, C., Montero-Ojeda, M., Bernardoni, A., Anderson, B.E., Parra, O., 1996. Human ehrlichiosis: report of the first case in Venezuela. Invest. Clin. 37, 35–49. Buller, R.S., Arens, M., Hmiel, S.P., Paddock, C.D., Sumner, J.W., Rikihisa, Y., Unver, Galdreault, K.E.E.N.E.R., Manian, F.A., Liddell, A.M., Schmulewitz, N., Storch, G.A., 1999. Ehrlichia ewingii recognized agent of human ehrlichiosis. N. Engl. J. Med. 341, 148–155. Calici, S.B., Galva˜o, M.A.M., Bacellar, F., Rocha, C.M.B.M., Mafra, C.L., Leite, R.C., Walker, D.H., 2004. Human ehrlichiosis in Brazil: first suspect cases. Braz. J. Infect. Dis. 8, 259– 262. Caspersen, K., Park, J.-H., Patil, S., Dumler, J.S., 2002. Genetic variability and stability of Anaplasma phagocytophila msp2 (p44). Infect. Immun. 70, 1230–1234.
266
R.Z. Machado et al. / Veterinary Parasitology 139 (2006) 262–266
Chae, J.S., Kim, M., Kim, M., Cho, Y., Park, B., 2003. Prevalence and sequence analyses of Neorickettsia risticii. Ann. NY. Acad. Sci. 990, 248–256. Dawson, J.E., Anderson, B.E., Fisbein, D.B., Sanchez, J.L., Goldsmith, C.S., Wilson, K.H., Duntley, C.W., 1991. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human Ehrlichiosis. J. Clin. Microbiol. 29, 2741–2745. Duarte, J.M.B., Merino, M.L., Gozales, M.S., Nunes, A.L.V., Garcia, J.M., Szabo´, M.J.P., Pandolfi, J.R., Arantes, I.G., Nascimento, A.A., Machado, R.Z.M., Araujo, J.P., Cata˜o-Dias, J.L., Werther, K., Garcia, J.E., Girio, J.S., Matushima, E.R., 2001. Order Artiodactyla family cervidae (Deer). Biology and medicine. In: Fowler, M.E., Cubas, Z.S. (Eds.), Biology, Medicine and Surgery of South American wild animals. Iowa State University Press, Ames, IA, pp. 402–422. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51, 2145–2165. Eng, T.R., Fishbein, D.B., Dawson, J.E., Greene, C.R., Redus, M., 1990. Survailence of human erhlichiosis in the United States. Ann. NY. Acad. Sci. 590, 306–307. Ewing, S.A., Dawson, J.E., Kocan, A.A., Barker, R.W., Warner, C.K., Panciera, R.J., Fox, J.C., Kocan, K.M., Blouin, E.F., 1995. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 32, 368–374. Galva˜o, M.A.M., Lamounier, J.A., Bonono, E., Tropia, M.S., Rezende, E.G., Calic, S.B., Chamone, C.B., Machado, M.C., Otoni, M.E.A., Leite, R.C., Caram, C., Mafra, C.L., Walker, D.H., 2002. Emerging and reemerging rickettsiosis in an endemic area of Minas Gerais State, Brazil. Cadernos de Sau´de Pu´blica 18, 1593–1597. Gongora-Biachi, R.A., Zavala-Velazquez, J., Castro-Sansores, C.J., Gonzalez-Martinez, P., 1999. First case of human ehrlichiosis in Mexico. E. I. D. 5, 481. Kocan, A., Levesque, G.C., Whitworth, L.C., Murphy, G.L., Ewing, S.A., Barker, R.W., 2000. Naturally occuring Ehrlichia chaffeensis infection in coyotes from Oklahoma. E. I. D. 6, 477–480. Kutler, K.L., 1994. Anaplasma infections in wild and domestic ruminants: a review. J. Wildl. Dis. 20, 12–20.
Lockhart, J.M., Davidson, D.E., Stallknecht, J.E., Dawson, J.E., Howerth, E.W., 1997. Isolation of Ehrlichia chaffeensis from wild white-tailed deer (Odocoileus virginianus) confirms their role as natural reservoir hosts. J. Clin. Microbiol. 35, 1681–1686. Massung, R., Slater, K., Owens, J.H., Nicholson, W.L., Mather, T.N., Solberg, V.B., Olson, J.G., 1998. Nested PCR assay for detection of Granulocytic Ehrlichiae. J. Clin. Microbiol. 36, 1090–1095. Paddock, C.D., Childs, J.E., 2003. Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin. Microbiol. Rev. 16, 37–64. Persing, D.H., 1996. PCR protocols for emerging infectious diseases. In: A supplement to Diagnostic Molecular Microbiology: Principles and Applications, ASM Press, Washington, DC. Ripoll, C.M., Remondegui, C.E., Ordonez, G., Arazamende, R., Fusaro, H., Ryman, M.J., Paddock, C.D., Zaki, S.R., Olson, J.G., Santos-Buch, C.A., 1999. Evidence of Rickettsial spotted fever and ehrlichial infections in a subtropical territory of Jujuy. Argentina Am. J. Trop. Med. Hyg. 61, 350–354. Szabo´, M.P.J., Labruna, M.B., Pereira, M.C., Duarte, J.M.B., 2003. Ticks (Acari:Ixodidade) in Wild Marsh Deer (Blastocerus dichotomus) from Southeast Brazil: Infestation before and after habitat loss. J. Med. Entomol. 40, 268–274. Torioni, E.S., Knowles, D.P., McGuire, T.C., Palmer, G.H., Suarez, C.E., Mcelwain, T.F., 1998. Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5. J. Clin. Microbiol. 36, 777–782. Unver, A., Rikihisa, Y., Ohashi, N., Cullman, L.C., Buller, R., Storch, G.A., 1999. Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and –negative human sera by using native and recombinant E. chaffeensis and E. canis antigens. J. Clin. Microbiol. 37, 3888– 3895. Unver, A., Felek, S., Paddock, N., Zhi, N., Horowitz, H.W., Wormser, G.P., Cullman, L.C., Rikihisa, Y., 2001. Western blot analysis of sera reactive to human monocytic ehrlichiosis and human granulocytic ehrlichiosis agents. J. Clin. Microbiol. 39, 3982–3986. Walker, D.H., Dumler, J.S., 1996. Emergence of the Ehrlichioses as Human Health Problems. E. I. D. 2, 18–29. Williams, C.V., Steenhouse, J.L.V., Bradley, J.M., Hancock, S.I., Hegarty, B.C., Breitschwerdt, E.B., 2002. Naturally occuring Ehrlichia chaffeensis infection in two prosimian primate species: Ring-tailed Lemurs (Lemur catta) and Ruffed Lemurs (Varecia variegata). E. I. D. 8, 1497–1500.