- Research article
- Open Access
Molecular characterization of Anaplasma platysstrains from dogs in Sicily, Italy
BMC Veterinary Researchvolume 2, Article number: 24 (2006)
The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes.
Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously.
At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.
The genus Anaplasma (Rickettsiales: Anaplasmataceae) contains obligate intracellular organisms found exclusively within membrane-bound inclusions or vacuoles in the cytoplasm of both vertebrate and invertebrate host cells [1, 2]. This genus includes pathogens of ruminants, A. marginale, A. centrale, A. bovis (formerly Ehrlichia bovis), and A. ovis. Also included in this genus is A. phagocytophilum (previously recognized as E. equi, E. phagocytophila and the human granulocytic ehrlichiosis (HGE) agent), which infects a wide range of hosts including humans and wild and domesticated animals, and A. platys (formerly E. platys) which is infective for dogs.
In dogs, A. platys develops within platelets and is the etiologic agent of canine infectious cyclic thrombocytopenia, but infected dogs are usually asymptomatic . Canine infections of A. platys have been reported throughout the world, including the United Sates [3–5], Spain [6, 7], France , Greece , Italy , Taiwan , China , Thailand , Japan [14–16], Venezuela [13, 17, 18], and Australia [19, 20]. However, A. platys infection is difficult to detect in vivo because the bacteremias are usually low [21–23]. Furthermore, serologic tests may be inaccurate because they are cross-reactive with other Anaplasma [8, 21, 24]. Recently, a PCR assay was optimized to allow for accurate identification of A. platys infection in dogs . The PCR test, confirmed by sequence analysis of amplicons, is considered to be the most reliable diagnostic test for A. platys to date.
Despite the worldwide distribution of A. platys, limited information is available on the genetic diversity of A. platys strains [13, 18, 25]. Herein, we characterized strains of A. platys from dogs in Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of 16S rDNA, heat shock operon groESL and the citrate synthase (gltA) genes.
Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy, for these studies. Blood was collected into sterile tubes with anticoagulant (EDTA), held at 4°C until arrival at the laboratory and then stored at -20°C for DNA extraction.
DNA extraction, PCR and sequence analysis
DNA was extracted from blood and tick samples using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, St. Louis, MO, USA). The A. marginale/A. centrale/A. ovis and A. phagocytophilum msp4 genes were amplified by PCR as reported previously [26, 27]. The Anaplasma spp. 16S rDNA was amplified by PCR using oligonucleotide primers 16SANA-F (5'-CAG AGT TTG ATC CTG GCT CAG AAC G-3') and 16SANA-R (5'-GAG TTT GCC GGG ACT TCT TCT GTA-3') as described previously [28, 29]. The A. platys-specific 16S rDNA, groESL and gltA PCRs were done as reported by Martin et al.  and Inokuma et al. , respectively. PCR reactions contained 2 μl (0.1–10 ng) DNA and 10 pmol of each primer in a 50-μl volume (1.5 mM MgSO4, 0.2 mM dNTP, 1X AMV/Tfl 5X reaction buffer, 5u Tfl DNA polymerase) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler (Eppendorf Mastercycler® personal, Westbury, NY, USA or Techne model TC-512, Cambridge, England, UK) for 35 cycles. Control reactions were done using the same procedures and reagents described above but without DNA added to the PCR reaction to rule out PCR contaminations. Carrying-over was ruled out due to the low number of PCR positive samples and the differences in the amplicon sequences. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb Plus DNA Ladder, Promega).
Amplified 16S rDNA, groESL and gltA fragments were resin purified (Wizard, Promega) and cloned into pGEM-T vector (Promega) for sequencing both strands by double-stranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University). At least two independent clones were sequenced. Multiple sequence alignment was performed using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD, USA) with an engine based on the Clustal W algorithm . BLAST  was used to search the NCBI databases to identify previously reported sequences with identity to those obtained in the study described herein.
Sequence accession numbers
The GenBank accession numbers for gltA sequences of A. platys strains are [GenBank: DQ525686–DQ525688].
Results and discussion
Prevalence of A. platysin dogs from Sicily
The observed prevalence of Anaplasma spp. was analyzed by PCR and sequence analysis of 16S rDNA amplicons. Of the 344 dogs analyzed, 14 (4%) were positive for Anaplasma spp. DNA (Table 1). Sequence analysis of 16S rDNA amplicons resulted in 100% identity to previously reported A. platys sequences. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA.
Previous 16S rDNA PCR-based A. platys studies in dogs reported observed prevalences of 33% (9/27, North Carolina, USA ), 32% (64/200, Okinawa, Japan ), 45% (10/22, Central Australia ) and 16% (7/43, Lara, Venezuela ). The analysis reported herein included a greater number of samples but the observed prevalence of A. platys in dogs was lower than in previous studies. These results suggested that A. platys infection in dogs in Sicily may be very low. However, differences in the sensitivity of the PCR due to the size of the amplicon and primer sequences may affect the results of prevalence studies reported by different groups.
Molecular characterization of A. platysstrains from Sicily
The 14 positive dog samples were characterized with A. platys-specific 16S rDNA, groESL and gltA PCR and sequence analysis. All dogs had 16S rDNA and groESL sequences identical to [GenBank: AY530806] and [GenBank: AY848753] A. platys Spanish and Italian strain sequences reported previously, respectively. These results agree with previous reports in which little genetic diversity was observed between 16S rDNA and groESL sequences of A. platys strains [16, 18, 20].
The sequence of A. platys gltA resulted in 3 different genotypes (Table 2). The sequences of A. platys from samples Miky, Dog 4 and Dog 9 were present in 9/14, 3/14 and 2/14 of the positive dog samples, respectively. A single nucleotide change in Dog 9 sequence resulted in an amino acid change (Table 2). Although the information about A. platys sequences is limited, these results suggest that gltA sequences may be more diverse than 16S rDNA and groESL sequences.
The result of gltA sequence analysis suggested that at least three different genotypes of A. platys infect dogs in Sicily. Two of the gltA sequences of Sicilian A. platys strains (Dog 4 and Dog 9) were different from sequences reported previously (Table 2). However, the gltA sequence of Miky strain was identical to the sequence of the Sommieres French strain. Huang et al.  suggested that A. platys strains are not geographically segregated. Although limited by the number of sequences available, the results of our study suggested that gltA may provide some phylogeographic information about A. platys strains. Nevertheless, although 16S rDNA, groESL and gltA sequences may be useful for phylogenetic studies of Anaplasma spp. , they were not informative for phylogenetic studies of A. platys strains.
The genetic diversity of A. marginale, A. phagocytophilum and A. ovis strains have been documented using different genetic markers [27, 29, 32, 33]. The results reported herein suggested that the genetic variation in A. platys may be similar to A. ovis but less than that observed in A. marginale and A. phagocytophilum. This low genetic variation may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum .
The tick vectors for the transmission of A. platys have not been extensively characterized. Rhipicephalus sanguineus [10, 34], Dermacentor auratus  and Hyalomma truncatum  have been suggested as possible vectors of A. platys. However, experimental transmission of A. platys with these tick species has not been demonstrated . Nevertheless, ticks of the genera Rhipicephalus, Dermacentor and Hyalomma are present in Sicily and could act as vectors of A. platys in this region .
Low observed prevalence (4%) of A. platys was detected in dogs from Sicily by PCR and sequence analysis of 16S rDNA, groESL and gltA genes. Three different gltA genotypes of A. platys were identified in these dogs. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis but lower than that for A. marginale and A. phagocytophilum. The lower genetic diversity of A. platys may have resulted from restricted movement of infected hosts as compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research to characterize genetic diversity of A. platys strains worldwide.
Dumler JS, Barbet AC, Bekker CPJ, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR: Reorganization of the 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 subjective synonyms of Ehrlichia phagocytophila. Int J Sys Evol Microbiol. 2001, 5: 12145-2165.
Kocan KM, de la Fuente J, Blouin EF, Garcia-Garcia JC: Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host-pathogen adaptations of a tick-borne rickettsia. Parasitol. 2004, 29: 285-300. 10.1017/S0031182003004700.
Harvey JW, Simpson CF, Gaskin JM: Cyclic thrombocytopenia induced by a Rickettsia-like agent in dogs. J Infect Dis. 1978, 137: 182-188.
Mathew JS, Ewing SA, Murphy GL, Kocan KM, Corstvet RE, Fox JC: Characterization of a new isolate of Ehrlichia platys (Order Rickettsiales) using electron microscopy and polymerase chain reaction. Vet Parasitol. 1997, 68: 1-10. 10.1016/S0304-4017(96)01052-7.
Kordick SK, Breitschwerdt EB, Hegarty BC, Southwick KL, Colitz CM, Hancock SI, Bradley JM, Rumbough R, Mcpherson JT, MacCormack JN: Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol. 1999, 37 (8): 2631-2638.
Sainz A, Amusategui I, Tesouro MA: Ehrlichia platys infection and disease in dogs in Spain. J Vet Diagn Invest. 1999, 11 (4): 382-384.
Solano-Gallego L, Llull J, Osso M, Hegarty B, Breitschwerdt E: A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet Res. 2006, 37 (2): 231-244. 10.1051/vetres:2005054.
Beaufils JP, Inokuma H, Martin-Granel J, Jumelle P, Barbault-Jumell M, Brouqui P: Anaplasma platys (Ehrlichia platys) infection in a dog in France: description of the case and characterization of the agent. Rev Med Vet. 2002, 153: 85-90.
Kontos VI, Papadopoulos O, French TW: Natural and experimental canine infections with a Greek strain of Ehrlichia platys. Vet Clin Pathol. 1991, 20: 101-105.
Sparagano OA, de Vos AP, Paoletti B, Camma C, de Santis P, Otranto D, Giangaspero A: Molecular detection of Anaplasma platys in dogs using polymerase chain reaction and reverse line blot hybridization. J Vet Diagn Invest. 2003, 15: 527-534.
Chang WL, Pan MJ: Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR. J Clin Microbiol. 1996, 34: 3142-3146.
Hua P, Yuhai M, Slide T, Yang S, Bohai W, Xiangrui C: Canine ehrlichiosis caused simultaneously by Ehrlichia canis and Ehrlichia platys. Microbiol Immunol. 2000, 44: 737-739.
Suksawat J, Pitulle C, Arraga-Alvarado C, Madrigal K, Hancock SI, Breitschwerdt EB: Coinfection with three Ehrlichia species in dogs from Thailand and Venezuela with emphasis on consideration of 16S ribosomal DNA secondary structure. J Clin Microbiol. 2001, 39 (1): 90-93. 10.1128/JCM.39.1.90-93.2001.
Inokuma H, Ohno K, Onishi T, Raoult D, Brouqui P: Detection of ehrlichial infection by PCR in dogs from Yamaguchi and Okinawa Prefecture. Jpn J Vet Med Sci. 2001, 63: 815-817. 10.1292/jvms.63.815.
Motoi Y, Satoh H, Inokuma H, Kiyuuna T, Muramatsu Y, Ueno H, Morita C: First detection of Ehrlichia platys in dogs and ticks in Okinawa, Japan. Microbiol Immunol. 2001, 45 (1): 89-91.
Unver A, Rikihisa Y, Kawahara M, Yamamoto S: Analysis of 16S rRNA gene sequences of Ehrlichia canis Anaplasma platys, and Wolbachia species from canine blood in Japan. Ann N Y Acad Sci. 2003, 990: 692-698.
Arraga-Alvarado C, Palmar M, Parra O, Salas P: Ehrlichia platys (Anaplasma platys) in dogs from Maracaibo, Venezuela: an ultrastructural study of experimental and natural infections. Vet Pathol. 2003, 40: 149-156. 10.1354/vp.40-2-149.
Huang H, Unver A, Perez MJ, Orellana NG, Rikihisa Y: Prevalence and molecular analisis of Anaplasma platys in dogs in Lara, Venezuela. Braz J Microbiol. 2005, 36: 211-216. 10.1590/S1517-83822005000300002.
Brown GK, Martin AR, Roberts TK, Aitken RJ: Detection of Ehrlichia platys in dogs in Australia. Aust Vet. 2001, 79: 554-558.
Martin AR, Brown GK, Dunstan RH, Roberts TK: Anaplasma platys: an improved PCR for its detection in dogs. Exp Parasitol. 2005, 109 (3): 176-180. 10.1016/j.exppara.2004.11.007.
French TW, Harvey JW: Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test. Am J Vet Res. 1983, 44: 2407-2411.
Bradford JF, Vore SJ, Prior WH: Ehrlichia platys infection in dogs. Lab Anim Sci. 1996, 46: 565-568.
Harrus S, Aroch I, Lavy E, Bark H: Clinical manifestations of infectious canine cyclic thrombocytopenia. Vet Rec. 1997, 141: 247-250.
Rikihisa Y: Diagnosis of emerging ehrlichial diseases of dogs, horses and humans. J Vet Intern Med. 2000, 14: 250-251. 10.1892/0891-6640(2000)14[250:EDOEED]2.0.CO;2.
Inokuma H, Fujii K, Okuda M, Onishi T, Beaufils JP, Raoult D, Brouqui P: Determination of the nucleotide sequences of heat shock operon groESL and the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys for phylogenetic and diagnostic studies. Clin Diagn Lab Immunol. 2002, 9 (5): 1132-1136. 10.1128/CDLI.9.5.1132-1136.2002.
de la Fuente J, Van Den Bussche RA, Garcia-Garcia JC, Rodríguez SD, García MA, Guglielmone AA, Mangold AJ, Friche Passos LM, Blouin EF, Kocan K: Phylogeography of New World strains of Anaplasma marginale (Rickettsiaceae: Ehrlichieae) based on major surface protein sequences. Vet Microbiol. 2002, 88: 275-285. 10.1016/S0378-1135(02)00122-0.
de la Fuente J, Massung RB, Wong SJ, Chu FK, Lutz H, Meli M, Mangold AJ, Kocan KM: Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains. J Clinic Microbiol. 2005, 43: 1309-1317. 10.1128/JCM.43.3.1309-1317.2005.
Stuen S, Nevland S, Moum T: Fatal cases of Tick-borne fever (TBF) in sheep caused by several 16S rRNA gene variants of Anaplasma phagocytophilum. Ann NY Acad Sci. 2003, 990: 433-434.
de la Fuente J, Naranjo V, Ruiz-Fons F, Höfle U, Fernández de Mera IG, Villanúa D, Almazán C, Torina A, Caracappa S, Kocan KM, Gortázar C: Potential vertebrate reservoir hosts and invertebrate vectors of Anaplasma marginale and A. phagocytophilum in central Spain. Vector-Borne Zoon Dis. 2005, 5: 390-401. 10.1089/vbz.2005.5.390.
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acid Res. 1994, 22: 4673-4680.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410. 10.1006/jmbi.1990.9999.
de la Fuente J, Lew A, Lutz H, Meli ML, Hofmann-Lehmann R, Shkap V, Molad T, Mangold AJ, Almazán C, Naranjo V, Gortázar C, Torina A, Caracappa S, García-Pérez AL, Barral M, Oporto B, Ceci L, Carelli G, Blouin EF, Kocan KM: Genetic diversity of Anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development. Anim Health Res Rev. 2005, 6: 75-89. 10.1079/AHR2005104.
de la Fuente J, Torina A, Caracappa S, Tumino G, Furlá R, Almazán C, Kocan KM: Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily. Vet Parasitol. 2005, 33: 357-362. 10.1016/j.vetpar.2005.05.063.
Inokuma H, Raoult D, Brouqui P: Detection of Ehrlichia platys DNA in brown dog ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan. J Clin Microbiol. 2000, 38: 4219-4221.
Parola P, Cornet JP, Sanogo YO, Miller RS, Thien HV, Gonzalez JP, Raoult D, Telford SR, Wongsrichanalai C: Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J Clin Microbiol. 2003, 41: 1600-1608. 10.1128/JCM.41.4.1600-1608.2003.
Simpson RM, Gaunt SD, Hair JA, Kocan KM, Henk WG, Casey HW: Evaluation of Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys. Am J Vet Res. 1991, 52: 1537-1541.
Torina A, Khoury C, Caracappa S, Maroli M: Ticks infesting livestock on farms in Western Sicily, Italy. Exp Appl Acarol. 2006, 38 (1): 75-86. 10.1007/s10493-005-5629-1.
This research was supported by the Ministry of Health, Italy, the Instituto de Ciencias de la Salud (ICS-JCCM), Spain (project 03052-00) and the Endowed Chair for Food Animal Research and the Oklahoma Agricultural Experiment Station Project 1669 (K.M. Kocan,). V. Naranjo is funded by Junta de Comunidades de Castilla – La Mancha (JCCM), Spain. Janet J. Rogers and Lisa Whitworth (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University) and Pilar Rubio (University of Alcalá de Henares, Spain) are acknowledged for DNA sequencing.
The author(s) declare that they have no competing interests.
José de la Fuente has designed and performed the sequence analyses and wrote the manuscript.
Alessandra Torina has organized and supervised the initial screening of dog samples by PCR.
Victoria Naranjo, Silviane Nicosia, Angelina Alongi and Francesco La Mantia have performed all the DNA extractions and PCR analyses.
Katherine M. Kocan has contributed to the final draft of the manuscript.
All authors have read and approved the final manuscript.