Molecular detection of Ehrlichia canisand Anaplasma platys in dogs in Southern Brazil

Detecção molecular de Ehrlichia canis eAnaplasma platys em cães do sul do Brasil

Camila Serina Lasta Andrea Pires dos Santos Joanne Belle Messick Simone Tostes Oliveira Alexander Welker Biondo Rafael Felipe da Costa Vieira Magnus Larruscaim Dalmolin Félix Hilario Diaz González About the authors

Abstracts

The aims of this study were to determine the occurrence ofAnaplasma platys and Ehrlichia canisinfection in dogs in Porto Alegre, Southern Brazil; and to investigate their association with hematological abnormalities. Serum samples from 196 dogs were first tested using dot-ELISA for antibodies against Anaplasmaspp. and Ehrlichia canis. Peripheral blood samples from 199 dogs were subjected to 16S rRNA nested PCR (nPCR) for A. platysand E. canis, followed by DNA sequencing to ensure pathogen identity. A total of 19/196 samples (9.69%) were positive forAnaplasma spp. using ELISA and 28/199 (14.07%) samples were positive for A. platys by nested PCR. All the dog samples were negative for E. canis, both in anti-E. canisantibody tests and in nested PCR. There were no significant differences in hematological parameters between A. platys-PCR positive and negative dogs and Anaplasma spp. serologically positive dogs, except for basophil counts, which were higher in nPCR-positive dogs. This is the first report showing A. platys presence in dogs in Southern Brazil. In conclusion, hematological parameters may not be sufficient to diagnose A. platys infection in dogs in Southern Brazil, probably due either to low pathogenicity or to chronic infection. On the other hand, E. canis may either have very low occurrence or be absent in dogs in Porto Alegre.

Anaplasmataceae; dogs; Southern Brazil; 16S rRNA gene


O objetivo deste estudo foi determinar a ocorrência deAnaplasma platys e Ehrlichia canis em cães de Porto Alegre, sul do Brasil, sua detecção molecular e associação com anormalidades hematológicas. Amostras séricas de 196 cães foram inicialmente triadas por dot-ELISA para a presença de anticorpos contraAnaplasma spp. e Ehrlichia canis. Amostras de sangue periférico de 199 cães foram submetidas à nested PCR (16S rRNA) paraA. platys e E. canis, seguido de sequenciamento do DNA para confirmar a identidade do agente. Do total, 19/196 (9,69%) amostras foram positivas para Anaplasma spp. por dot-ELISA e 28/199 (14,07%) por nPCR. Todas as amostras dos cães foram negativas para E. canis no teste sorológico anti-E. canis e também na nPCR. Não houve diferença significativa nos parâmetros hematológicos, exceto a contagem de basófilos, que apresentou valores mais altos em cães positivos na nPCR para A. platys. Este é o primeiro relato da presença de A. platys no Rio Grande do Sul, e a primeira detecção molecular do agente no sul do Brasil. Em conclusão, parâmetros hematológicos não são suficientes para diagnosticar a infecção porA. platys em cães, provavelmente devido sua baixa patogenicidade ou infecção crônica. Por outro lado, E. canisparece ter ocorrência baixa ou mesmo nula em cães de Porto Alegre.

Anaplasmataceae; cães; sul do Brasil; gene 16S rRNA


Introduction

Anaplasma platys and Ehrlichia canis are tick-borne bacteria that cause mild to severe disease in dogs (BEALL et al., 2008Beall MJ, Chandrashekar R, Eberts MD, Cyr KE, Diniz PPVP, Mainville C, et al. Serological and Molecular Prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum and Ehrlichia Species in Dogs from Minnesota. Vector Borne Zoonotic Dis 2008; 8(4): 455-464. http://dx.doi.org/10.1089/vbz.2007.0236
http://dx.doi.org/10.1089/vbz.2007.0236...
; GAUNT et al., 2010Gaunt SD, Beall MJ, Stillman BA, Lorentzen L, Diniz PPVP, Chandrashekar R, Breitschwerdt EB. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasites & Vectors 2010; 3:33. PMid:20377870 PMCid:PMC2859368. http://dx.doi.org/10.1186/1756-3305-3-33
http://dx.doi.org/10.1186/1756-3305-3-33...
). Both organisms are transmitted by ticks, mainly byRhipicephalus sanguineus. Anaplasma platys - formerly known as Ehrlichia platys (DUMLER et al., 2001) - andE. canis, from the Anaplasmataceae family, cause canine infectious cyclic thrombocytopenia (CICT) and canine monocytic ehrlichiosis (CME), respectively (HARVEY, 2006Harvey JW. Thrombocytitropic Anaplasmosis (A. platys [E. platys] Infection). In: Greene CE. Infectious Diseases of the Dog and Cat. 3rd ed. St. Louis: Elsevier; 2006. p. 229-231.; DANTAS-TORRES, 2008Dantas-Torres F. Canine vector-borne diseases in Brazil. Parasit Vectors 2008; 1: 25. http://dx.doi.org/10.1186/1756-3305-1-25
http://dx.doi.org/10.1186/1756-3305-1-25...
).

Anaplasma platys develops inside canine platelets, with no clearly established pathogenic role in dogs (AGUIRRE et al., 2006Aguirre E, Tesouro MA, Ruiz L, Amusategui I, Sainz A. Genetic Characterization of Anaplasma (Ehrlichia) platys in Dogs in Spain. J Vet Med B Infect Dis Vet Public Health 2006; 53(4): 197-200. PMid:16629989. http://dx.doi.org/10.1111/j.1439-0450.2006.00937.x
http://dx.doi.org/10.1111/j.1439-0450.20...
; HARVEY, 2006Harvey JW. Thrombocytitropic Anaplasmosis (A. platys [E. platys] Infection). In: Greene CE. Infectious Diseases of the Dog and Cat. 3rd ed. St. Louis: Elsevier; 2006. p. 229-231.). Reports on experimental and natural infections have mostly indicated that A. platys causes few or no clinical signs in dogs in the United States (HARVEY, 2006Harvey JW. Thrombocytitropic Anaplasmosis (A. platys [E. platys] Infection). In: Greene CE. Infectious Diseases of the Dog and Cat. 3rd ed. St. Louis: Elsevier; 2006. p. 229-231.; GAUNT et al., 2010Gaunt SD, Beall MJ, Stillman BA, Lorentzen L, Diniz PPVP, Chandrashekar R, Breitschwerdt EB. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasites & Vectors 2010; 3:33. PMid:20377870 PMCid:PMC2859368. http://dx.doi.org/10.1186/1756-3305-3-33
http://dx.doi.org/10.1186/1756-3305-3-33...
). However, A. platysstrains are reportedly more virulent in France, Spain, Turkey and Tunisia (BEAUFILS et al., 2002Beaufils JP, Inokuma H, Martin-Granel J, Jumelle P, Barbault-Jumelle 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(2): 85-90.; AGUIRRE et al., 2006Aguirre E, Tesouro MA, Ruiz L, Amusategui I, Sainz A. Genetic Characterization of Anaplasma (Ehrlichia) platys in Dogs in Spain. J Vet Med B Infect Dis Vet Public Health 2006; 53(4): 197-200. PMid:16629989. http://dx.doi.org/10.1111/j.1439-0450.2006.00937.x
http://dx.doi.org/10.1111/j.1439-0450.20...
; ULUTAS et al., 2007Ulutas B, Bayramli G, Karagenç T. First Case of Anaplasma (Ehrlichia) platys Infection in a Dog in Turkey. Turk J Vet Anim Sci 2007; 31(4): 279-282., M'GHIRBI et al., 2009M'ghirbi Y, Ghorbel A, Amouri M, Nebaoui A, Haddad, Bouattour A. Clinical, serological, and molecular evidence of ehrlichiosis and anaplasmosis in dogs in Tunisia. Parasitol Res 2009; 104(4): 767-774. http://dx.doi.org/10.1007/s00436-008-1253-4
http://dx.doi.org/10.1007/s00436-008-125...
), with clinical signs that are probably influenced by stress, immune status, breed predisposition (AGUIRRE et al., 2006Aguirre E, Tesouro MA, Ruiz L, Amusategui I, Sainz A. Genetic Characterization of Anaplasma (Ehrlichia) platys in Dogs in Spain. J Vet Med B Infect Dis Vet Public Health 2006; 53(4): 197-200. PMid:16629989. http://dx.doi.org/10.1111/j.1439-0450.2006.00937.x
http://dx.doi.org/10.1111/j.1439-0450.20...
) or strain variability. In contrast, E. canis infects monocytes and has been associated with serious and sometimes fatal disease in dogs (NEER; HARRUS, 2006Neer MT, Harrus S. Canine monocytotropic ehrlichiosis and neorickettsiosis (E. canis, E. chaffeensis, E. ruminantium, N. sennetsu, and N. risticii infections). In: Greene CE. Infectious Diseases of the Dog and Cat. 3rd ed. St. Louis: Elsevier; 2006. p. 203-216.). It has also been suggested to be a zoonotic agent in Venezuela (PEREZ et al., 1996Perez M, Rikihisa Y, Wen B. Ehrlichia canis-like agent isolated from a man in Venezuela: antigenic and genetic characterization. J Clin Microbiol 1996; 34(9): 2133-2139. PMid:8862572 PMCid:PMC229204., 2006Perez M, Bodor M, Zhang C, Xiong Q, Rikihisa Y. Human infection with Ehrlichia canis accompanied by clinical signs in Venezuela. Ann N Y Acad Sci 2006; 1078: 110-117. PMid:17114689. http://dx.doi.org/10.1196/annals.1374.016
http://dx.doi.org/10.1196/annals.1374.01...
).

No serological surveys on A. platys have been performed to date in Brazil, whereas the seroprevalence of E. canis in dogs in Brazil may range from 0.7 to 92.3%, depending on the population, geographical area and diagnostic test used (OLIVEIRA et al., 2000Oliveira D, Tie Nishimori C, Costa MT, Machado RZ, Castro MB. Anti- Ehrlichia canis antibodies detection by “Dot-ELISA” in naturally infected dogs. Rev Bras Parasitol Vet 2000; 9(1): 1-5.; LABARTHE et al., 2003Labarthe N, Pereira MC, Barbarini O, McKee W, Coimbra CA, Hoskins J. Serologic Prevalence of Dirofilaria immitis, Ehrlichia canis, and Borrelia burgdorferi Infections in Brazil. Vet Ther 2003; 4(1): 67-75. PMid:12756637.; VIEIRA et al., 2011Vieira RFC, Biondo AW, Guimarães AMS, Santos AP, Santos RP, Dutra LH, et al. Ehrlichiosis in Brazil. Rev Bras Parasitol Vet 2011; 20(1): 1-12. PMid:21439224. http://dx.doi.org/10.1590/S1984-29612011000100002
http://dx.doi.org/10.1590/S1984-29612011...
). Moreover, molecular detection of A. platys and E. canis in dogs has been conducted in Brazil and has shown widely variable prevalence, from 7.8 to 88% and from 8.1 to 55%, respectively (DAGNONE et al., 2003Dagnone AS, Morais HSA, Vidotto MC, Jojima FS, Vidotto O. Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a Hospital population in South Brazil. Vet Parasitol 2003; 117(4): 285-290. PMid:14637030. http://dx.doi.org/10.1016/j.vetpar.2003.10.001
http://dx.doi.org/10.1016/j.vetpar.2003....
, 2009Dagnone AS, Souza AI, André MR, Machado RZ. Molecular diagnosis of Anaplasmataceae organisms in dogs with clinical and microscopical signs of ehrlichiosis. Rev Bras Parasitol Vet 2009; 18(4): 20-25. PMid:20040204. http://dx.doi.org/10.4322/rbpv.01804004
http://dx.doi.org/10.4322/rbpv.01804004...
; COSTA Jr., 2007b; RAMOS et al., 2009; SANTOS et al. 2009aSantos F, Coppede JS, Pereira ALA, Oliveira LP, Roberto PG, Benedetti RBR, et al. Molecular evaluation of the incidence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs from Ribeirão Preto, Brazil. Vet J 2009a; 179(1): 145-148. PMid:17920967. http://dx.doi.org/10.1016/j.tvjl.2007.08.017
http://dx.doi.org/10.1016/j.tvjl.2007.08...
).

Although R. sanguineus is a commonly found tick species in Southern Brazil (RIBEIRO et al., 1997Ribeiro VLS, Weber MA, Fetzer LO, Vargas CRB. Espécies e prevalência das infestações por carrapatos em cães de rua da cidade de Porto Alegre, RS, Brasil. Cienc Rural 1997; 27(2): 285-289.http://dx.doi.org/10.1590/S0103-84781997000200019
http://dx.doi.org/10.1590/S0103-84781997...
), it has not yet been fully established whether A. platys and E. canis cause infection in dogs in the state of Rio Grande do Sul. Accordingly, the aims of the present study were to determine the serological and molecular prevalence of A. platys and E. canis and to correlate infection with hematological abnormalities in two populations of naturally infected dogs in Porto Alegre, the capital of Rio Grande do Sul, Southern Brazil.

Materials and Methods

Study population and samples

This study was carried out in the city of Porto Alegre (30° 01′ 59″ S and 51° 13′ 48″ W), the capital of the state of Rio Grande do Sul, Southern Brazil, with an estimated population of 1,400,000 inhabitants. Blood samples were collected between May 2007 and February 2009, from 53 stray dogs at the city's Zoonosis Control Center and 146 semi-owned dogs (dogs that were living outdoors, with full access to the streets, i.e. free roaming) on a low-income island (Arquipelago district) within the city limits, thus totaling 199 dogs. Only one sample was taken from each dog.

The Arquipelago district is the biggest in Porto Alegre and is formed by 15 islands. Its population is approximately 5,000 inhabitants and most of them rely on garbage recycling. The Zoonosis Control Center did not have precise information about where each dog was initially caught, but it functions throughout the city. The data recorded on each animal included information on breed, age and location. In the Arquipelago district, the dogs were selected by convenience, at times when clients volunteered their dogs during attendance campaigns, without specific inclusion criteria. Only the animals that were receiving antibiotic therapy were excluded from the study. This work was approved by the Ethics Committee for Animal Experimentation and Animal Welfare of Universidade Federal do Rio Grande do Sul (UFRGS) (number 13.313).

Blood collection and hematological analysis

Blood samples were collected and aliquoted into EDTA tubes for hematological analyses and PCR, and into serum tubes for serological tests. Samples from three dogs had insufficient volume for serum tests. Blood cell counts were determined as previously described (RIZZI et al., 2010). Blood smears were examined microscopically for the presence of morulae, and the peripheral blood smear WBC differential was ascertained by counting 100 leukocytes. This was done by a person who was blinded to the serological and nPCR results.

Serological test: dot-ELISA-based assay

Samples were tested for antibody reactivity against E. canis and Anaplasma spp. using a commercial rapid in-clinic ELISA assay (SNAP 4Dx® test kit, IDEXX Laboratories, Inc., Westbrook, Maine, USA), according to the manufacturer's instructions.

DNA extraction, nested PCR and DNA sequencing

DNA was isolated from 200 µL of EDTA blood using the QIAamp DNA Blood Mini kit (Qiagen, Valencia, California, USA), following the manufacturer's instructions. Negative control purifications using ultra-pure water were performed to monitor cross-contamination for each batch of 10 samples. To verify the existence of amplifiable DNA in the samples, a PCR assay for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as previously described (SANTOS et al., 2009bSantos AP, Messick JB, Biondo AW, Oliveira ST, Pedralli V, Lasta CS, et al. Design, optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil. Vet Clin Pathol 2009b; 38(4): 443-452. http://dx.doi.org/10.1111/j.1939-165X.2009.00158.x
http://dx.doi.org/10.1111/j.1939-165X.20...
).

Samples were initially screened using the universal primer fD1 and the genus-specific primer EHR16SR (INOKUMA et al., 2001Inokuma H, Ohno K, Onishi T, Raoult D, Brouqui P. Detection of Ehrlichial Infection by PCR in dogs from Yamaguchi and Okinawa Prefectures, Japan. J Vet Med Sci 2001; 63(7): 815-817. PMid:11503912. http://dx.doi.org/10.1292/jvms.63.815
http://dx.doi.org/10.1292/jvms.63.815...
). These primers amplify 760 bp of the partial sequence of the 16S rRNA gene of Ehrlichia and Anaplasmaspecies. Briefly, 5 µL of DNA was used as a template for the primary amplification, in a total reaction mixture of 25 µL containing 1.5 mM of MgCl2, 0.2 mM of each deoxynucleoside triphosphate (dNTP), 0.25 U of Taq polymerase (Go Taq Fexi Promega, Madison, WI, USA) and 0.1 mM of each primer. After initial denaturation at 94 °C for 1 min, the amplification consisted of 35 cycles of 1 min each at temperatures of 94 °C, 55 °C and 72 °C for denaturation, annealing and extension, respectively.

The second amplification (nested PCR) was carried out using the primers CANIS and GA1UR for E. canis (504 bp), and PLATYS F and PLATYS R for A. platys (408 bp) with a few modifications (INOKUMA et al., 2001Inokuma H, Ohno K, Onishi T, Raoult D, Brouqui P. Detection of Ehrlichial Infection by PCR in dogs from Yamaguchi and Okinawa Prefectures, Japan. J Vet Med Sci 2001; 63(7): 815-817. PMid:11503912. http://dx.doi.org/10.1292/jvms.63.815
http://dx.doi.org/10.1292/jvms.63.815...
), which included: 1) PCR products were diluted 1:5 with nuclease-free water; and 2) 1 µL of the solution was used as the template DNA for the second reaction. The conditions for the PCR amplification were the same as for the first round of PCR, except for the annealing temperature for E. canis (57.6 °C) and the number of cycles (39). The positive controls were 10-fold dilutions of a plasmid containing starting concentrations of 5.47 × 109 and 6.18 × 1010 copies/µL of the 16S rRNA gene of E. canisand A. platys, respectively, until reaching less than 1 copy/µL. In each PCR run, the negative control consisted of all reagents (excluding sample) and ultra-pure water. The negative control from the first PCR run was also used as the negative control in the second run. Positive controls, including the control with the lowest copy number (determined as the detection limit for each assay) were included in every run.

The amplified PCR products were subjected to gel electrophoresis in 1.5% agarose gels for one hour at 100 V, followed by ethidium bromide staining (1 µg/mL), and were viewed under a 312 nm UV light transilluminator. The gels were subsequently photographed using Epi Chem II Darkroom® (UVP, Inc., Upland, California, USA). In order to minimize potential risks of contamination, DNA extractions, PCR preparation, PCR amplification, and agarose gel electrophoresis were performed in separate rooms.

All the nPCR products were purified using the QIAprep Spin Miniprep kit (QIAGEN), and the amplicons were directly sequenced with both forward and reverse primers (Purdue Genomics Core Facility, Purdue University, West Lafayette, Indiana, USA). Primers were deleted from DNA sequences obtained from the Anaplasma platys 16S rRNA gene and were compared with those of the GenBank® database using the BLAST® nucleotide (ALTSCHUL et al., 1990Altschul SF, Gish W, Miller WE, Myers W, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215(3): 403-410. PMid:2231712.) in order to search for identicalness.

Positive controls

Positive controls were obtained from dogs naturally infected byE. canis and A. platys in the city of Londrina, state of Paraná, Southern Brazil. To construct reliable positive controls, nPCR for E. canis and A. platysdetection was performed as described above. Amplicons (504 bp fragment of the 16S rRNA gene for E. canis and 408 bp fragment for A. platys) were purified from gel (Zymoclean DNA Gel Recovery, Zymo Research, Orange, California, USA), and were cloned into the pGEM-T Easy Vector (Promega, Madison, Wisconsin, USA) followed by transformation in JM 109 Competent Cells (Promega, Madison, Wisconsin, USA). Plasmids with inserts were isolated, grown and purified using a commercial kit (Miniprep, QIAGEN, Valencia, California, USA).

The DNA concentration of positive controls was quantified by means of scanning UV spectrophotometry (NanoDrop® ND-1000 UV/Vis Spectrophotometer, Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) to determine the number of copies/µL. The detection limit for the nPCR was determined by using serial 10-fold dilutions of the positive plasmid controls, spiked in Herring Sperm DNA (KPL, Gaithersburg, Maryland, USA) as the DNA template.

Statistical analysis

Statistical univariate analysis on associations between seropositivity and PCR results and hematological parameters was conducted using the chi-square test. Fisher's exact test was used to evaluate associations between seropositivity and nPCR positivity status when the expected frequency was less than five, using the Stata 11.1 software (Stata Corp, College Station, Texas, USA). Statistical significance was defined as p < 0.05.

Results

The nPCR assays were able to amplify the control template diluted to as few as 101 and 103 gene copies per microliters for E. canis and A. platys, respectively, and all the samples were positive for GAPDH, thereby confirming the presence of amplifiable DNA in the samples.

All the samples were negative for the presence of antibodies forE. canis and E. canis 16S rRNA DNA. Among the dog serum samples, 19/196 (9.69%) showed antibodies that were reactive toAnaplasma spp. using the SNAP 4Dx® test. A. platys DNA (16S rRNA gene) was amplified by nPCR in 28/199 dogs (14.07%). There was no positive association between the PCR and ELISA results (p > 0.05).

Among the 28 A. platys nPCR-positive dogs, 7 (n = 53) were from the dogs from the city's Zoonosis Control Center (urban area) and 21 (n = 146) from the Arquipelago district (suburban area). No statistical difference between PCR-positivity and location of the dogs was observed (p-value = 0.448). Nineteen (67.8%) of the nPCR positive dogs were anemic (RBC < 5.5 × 106/µL) and 16 (61.5%) were thrombocytopenic (platelets < 200 × 103/µL) (Table 1). Two positive samples had fibrin-clot formation and the data from these samples were not included in the analyses. There was no association between a positive serological test or PCR and other CBC abnormalities except for the basophile count, which was higher the reference value for the leukocyte differential in PCR-positive dogs (p = 0.015).

Table 1.
Anemia and thrombocytopenia associated with positive PCR forAnaplasma platys or serology forAnaplasma spp. from dogs.

The A. platys 16S rRNA gene partial sequences obtained in this study were 100% identical to each other. A partial 16S rRNA gene sequence of 408 bp, representative of all samples, was deposited in GenBank® database as the Porto Alegre isolate, under the accession number JF418996. Sequence comparisons on the 408 bp fragment from Porto Alegre revealed that the amplicon sequences were identical (100%) to A. platys samples from other Brazilian areas such as Campo Grande/MS/Center-West Brazil (JX118826) and Ribeirão Preto/SP/Southeast Brazil (EF052622), and from other countries: Philippines (Q8947792), Cape Verde (GQ395385), Croatia (JQ396431), Italy (EU439943), Malaysia (JF683610), Portugal (EU004823), Spain (AY530806), Japan (AF288136), Venezuela (HE856819) and Thailand (EF13945). The lowest identity (99.75%) was with anA. platys sample identified in dogs in Venezuela (AF399917). A lower sequence identity of 98% was found in relation to a German sample ofA. phagocytophilum (HM480383).

Discussion

All the samples tested negative for E. canis by PCR and the SNAP 4Dx® test, thus suggesting that there was lower occurrence ofE. canis in Porto Alegre than in other Brazilian regions. A previous study in Southern Brazil using serological tests showed low occurrence ofE. canis in asymptomatic dogs, of 4.8% (SAITO et al., 2008Saito TB, Cunha-Filho NA, Pacheco RC, Ferreira F, Pappen FG, Farias NAR, et al. Canine Infection by Rickettsiae and Ehrlichiae in Southern Brazil. Am J Trop Med Hyg 2008; 79(1): 102-108. PMid:18606772.). However, higher prevalence was found in other Brazilian regions, ranging from 24.8% to 44.7%, using similar populations of dogs (AGUIAR et al., 2007; COSTA Jr et al., 2007a, SILVA et al., 2010Silva JN, Almeida ABPF, Sorte ECB, Freitas AG, Santos LGF, Aguiar DM, et al. Soroprevalência de anticorpos anti-Ehrlichia canis em cães de Cuiabá, Mato Grosso. Rev Bras Parasitol Vet 2010; 19(2): 108-111. PMid:20624348. http://dx.doi.org/10.4322/rbpv.01902008
http://dx.doi.org/10.4322/rbpv.01902008...
; SOUZA et al., 2010Souza BMP, Leal DC, Barboza DCPM, Uzêda RS, Alcântara AC, Ferreira F, et al. Prevalence of ehrlichial infection among dogs and ticks in Northeastern Brazil. Rev Bras Parasitol Vet 2010; 19(2): 89-93. PMid:20624344. http://dx.doi.org/10.4322/rbpv.01902004
http://dx.doi.org/10.4322/rbpv.01902004...
). Although the tick vector R. sanguineus is abundant throughout all urban areas of Brazil (SAITO et al., 2008Saito TB, Cunha-Filho NA, Pacheco RC, Ferreira F, Pappen FG, Farias NAR, et al. Canine Infection by Rickettsiae and Ehrlichiae in Southern Brazil. Am J Trop Med Hyg 2008; 79(1): 102-108. PMid:18606772.), including in the state of Rio Grande do Sul (RIBEIRO et al., 1997Ribeiro VLS, Weber MA, Fetzer LO, Vargas CRB. Espécies e prevalência das infestações por carrapatos em cães de rua da cidade de Porto Alegre, RS, Brasil. Cienc Rural 1997; 27(2): 285-289.http://dx.doi.org/10.1590/S0103-84781997000200019
http://dx.doi.org/10.1590/S0103-84781997...
), it is important to consider that different populations of this tick species are found in Brazil (MORAES-FILHO et al., 2011Moraes-Filho J, Marcili A, Nieri-Bastos FA, Richtzenhain LJ, Labruna MB. Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop 2011; 117(1): 51-55. PMid:20858451. http://dx.doi.org/10.1016/j.actatropica.2010.09.006
http://dx.doi.org/10.1016/j.actatropica....
). Thus, the negative results for E. canis found in the present study may have been due to differences in the vector competence of the R. sanguineus (SZABÓ et al., 2005Szabó MP, Mangold AJ, João CF, Bechara GH, Guglielmone AA. Biological and DNA evidence of two dissimilar populations of the Rhipicephalus sanguineus tick group (Acari: Ixodidae) in South America. Vet Parasitol 2005; 130(1-2): 131-140. PMid:15893080. http://dx.doi.org/10.1016/j.vetpar.2005.03.008
http://dx.doi.org/10.1016/j.vetpar.2005....
). Although peripheral blood samples were used in this study, it may be postulated that false-negative results from nPCR for E. canis detection occurred because the agent could be sequestered in the spleen and bone marrow during the subclinical or chronic phase of the disease (MYLONAKIS et al., 2003Mylonakis ME, Koutinas AF, Billinis C, Leontides LS, Kontos V, Papadopoulos O, et al. Evaluation of cytology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): a comparison between five methods. Vet Microbiol 2003; 91(2-3):197-204. http://dx.doi.org/10.1016/S0378-1135(02)00298-5
http://dx.doi.org/10.1016/S0378-1135(02)...
). However, we did not find any seropositive (exposed) dog, which leads us to conclude that occurrence of E. canis in dogs in this work is very low or absent.

Six dogs showed positive results in both tests (Table 2). Thirteen dogs (13/19, 52.6%) that were serologically positive for Anaplasma sp. were negative according to nPCR, which suggests that there was a likelihood of previous exposure to the agent. Also, the cyclic parasitemia in dogs infected with A. platys may explain this discrepancy between serological and molecular results, as previously observed (FERREIRA et al., 2007Ferreira RF, Cerqueira AMF, Pereira AM, Guimarães CM, Sá AG, Abreu FS, et al. Anaplasma platys Diagnosis in Dogs: Comparison Between Morphological and Molecular Tests. Intern J Appl Res Vet Med 2007; 5(3): 113-119., 2008bFerreira RF, Cerqueira AMF, Pereira AM, Ferreira MS, Almosny NRP. Hematologic Parameters in Polymerase Chain Reaction-Positive and -Negative Dogs for Anaplasma platys Presenting Platelet Inclusion Bodies. Intern J Appl Res Vet Med 2008b; 6(3): 185-190.). Although A. phagocytophilum antigens are used in the commercial SNAP 4Dx® test kit, A. platysantibodies cross-react with the A. phagocytophilum spot in this test (CHANDRASHEKAR, et al., 2010Chandrashekar R, Mainville CA, Beall MJ, O'Connor T, Eberts MD, Alleman RA, et al. Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs. Am J Vet Res 2010; 71(12): 1443-1450. PMid:21117995. http://dx.doi.org/10.2460/ajvr.71.12.1443
http://dx.doi.org/10.2460/ajvr.71.12.144...
). The kit uses a synthetic peptide based on p44 A. phagocytophilumimmunodominant protein and detects IgM and IgG antibodies. Because A. phagocytophilum and A. platys are closely related and share epitopes, the kit can be used for A. platys diagnosis (FERREIRA et al., 2008a.) On the other hand, many dogs (19/25, 76%) that were positive by Anaplasma sp. nPCR were negative by serological tests for this agent (Table 2), which suggest that negative serological findings do not imply absence of organism infection (FERREIRA et al., 2008a). Positive Anaplasma sp. nPCR results with negative serological results might also indicate an early stage of infection.Anaplasma sp. nPCR was able to detect 103 gene copies per reaction, but it is also possible that dogs have lower numbers of copies of A. platys DNA, thus resulting in false negative results.

Table 2.
Anaplasma spp. serology and Anaplasma platys nPCR results in dogs.

The possibility that dogs in our study were infected or co-infected withA. phagocytophilum cannot be ruled out, since dogs may be asymptomatic carriers for both agents. In a survey of zoonotic vector-borne diseases conducted previously in Botucatu, state of São Paulo, 198 sick dogs with clinical signs of tick-borne disease showed no evidence of A. phagocytophilum exposure or infection (DINIZ et al., 2007Diniz PPVP, Schwartz DS, De Morais HSA, Breitschwerdt EB. Surveillance for zoonotic vector-borne infections using sick dogs from southeastern Brazil. Vector Borne Zoonotic Dis 2007; 7(4): 689-698. PMid:18021025. http://dx.doi.org/10.1089/vbz.2007.0129
http://dx.doi.org/10.1089/vbz.2007.0129...
). In fact, A. phagocytophilum was recently detected in dogs by real-time PCR, in a survey conducted in the state of Rio de Janeiro (SANTOS et al., 2011Santos HA, Pires MS, Vilela JA, Santos TM, Faccini JL, Baldani CD, et al. Detection of Anaplasma phagocytophilum in Brazilian dogs by real-time polymerase chain reaction. Journal of Veterinary Diagnostic Investigation 2011; 23(4): 770-774. PMid:21908321. http://dx.doi.org/10.1177/1040638711406974
http://dx.doi.org/10.1177/10406387114069...
), and in wild birds in the states of São Paulo and Goiás (MACHADO et al., 2012Machado RZ, Andre MR, Werther K, Sousa E, Gavioli FA, Junior JFRA. Migratory and Carnivorous Birds in Brazil: Reservoirs for Anaplasma and Ehrlichia Species? Vector Borne Zoonotic Diseases 2012; 12(8): 705-708. http://dx.doi.org/10.1089/vbz.2011.0803
http://dx.doi.org/10.1089/vbz.2011.0803...
). Moreover, new genotypes of Anaplasmataceae agents have been reported in wild animals in Brazil (ANDRÉ et al., 2010André MR, Adania CH, Machado RZ, Allegretti SM, Felippe PAN, Silva KF, et al. Molecular and Serologic Detection of Ehrlichia spp. in Endangered Brazilian Wild Captive Felids. J Wildl Dis 2010; 46(3): 1017-1023. PMid:20688716., 2012André MR, Dumler JS, Scorpio DG, Teixeira RH, Allegretti SM, Machado RZ. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks Tick Borne Dis 2012; 3(4): 247-253. http://dx.doi.org/10.1016/j.ttbdis.2012.04.002
http://dx.doi.org/10.1016/j.ttbdis.2012....
; SACCHI et al., 2012Sacchi AB, Duarte JM, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comp Immunol Microbiol Infect Dis 2012; 35(4): 325-334. http://dx.doi.org/10.1016/j.ttbdis.2012.04.002
http://dx.doi.org/10.1016/j.ttbdis.2012....
).

Although dogs in Porto Alegre were infected by A. platysin the same way as described in other Brazilian regions (FERREIRA et al., 2007Ferreira RF, Cerqueira AMF, Pereira AM, Guimarães CM, Sá AG, Abreu FS, et al. Anaplasma platys Diagnosis in Dogs: Comparison Between Morphological and Molecular Tests. Intern J Appl Res Vet Med 2007; 5(3): 113-119., 2008bFerreira RF, Cerqueira AMF, Pereira AM, Ferreira MS, Almosny NRP. Hematologic Parameters in Polymerase Chain Reaction-Positive and -Negative Dogs for Anaplasma platys Presenting Platelet Inclusion Bodies. Intern J Appl Res Vet Med 2008b; 6(3): 185-190.; COSTA Jr, 2007b; DAGNONE et al., 2009Dagnone AS, Souza AI, André MR, Machado RZ. Molecular diagnosis of Anaplasmataceae organisms in dogs with clinical and microscopical signs of ehrlichiosis. Rev Bras Parasitol Vet 2009; 18(4): 20-25. PMid:20040204. http://dx.doi.org/10.4322/rbpv.01804004
http://dx.doi.org/10.4322/rbpv.01804004...
; RAMOS et al., 2009), only two isolates have been molecularly characterized to date in Brazil (CARDOZO et al., 2007Cardozo GP, Oliveira LP, Zissou VG, Donini IAN, Roberto PG, Marins M. Analysis of the 16S rRNA gene of Anaplasma platys detected in dogs from Brazil. Braz J Microbiol 2007; 38(3): 478-479. http://dx.doi.org/10.1590/S1517-83822007000300017
http://dx.doi.org/10.1590/S1517-83822007...
). Sequence comparisons on the partial 16S rRNA gene in the samples from dogs in Porto Alegre revealed that the sequences were identical (100%) to those from other locations in Brazil (Campo Grande/MS/ Center-West Brazil and Ribeirão Preto/SP/Southeast Brazil), as well as to those from other strains around the world (INOKUMA et al., 2000Inokuma 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(11): 4219-4221. PMid:11060094 PMCid:PMC87567., 2001Inokuma H, Ohno K, Onishi T, Raoult D, Brouqui P. Detection of Ehrlichial Infection by PCR in dogs from Yamaguchi and Okinawa Prefectures, Japan. J Vet Med Sci 2001; 63(7): 815-817. PMid:11503912. http://dx.doi.org/10.1292/jvms.63.815
http://dx.doi.org/10.1292/jvms.63.815...
; AGUIRRE et al., 2006Aguirre E, Tesouro MA, Ruiz L, Amusategui I, Sainz A. Genetic Characterization of Anaplasma (Ehrlichia) platys in Dogs in Spain. J Vet Med B Infect Dis Vet Public Health 2006; 53(4): 197-200. PMid:16629989. http://dx.doi.org/10.1111/j.1439-0450.2006.00937.x
http://dx.doi.org/10.1111/j.1439-0450.20...
; PINYOOWONG et al., 2008Pinyoowong D, Jittapalapong S, Suksawat F, Stich RW, Thamchaipenet A. Molecular characterization of Thai Ehrlichia canis and Anaplasma platys strains detected in dogs. Infect Genet Evol 2008; 8(4): 433-438. PMid:17643354. http://dx.doi.org/10.1016/j.meegid.2007.06.002
http://dx.doi.org/10.1016/j.meegid.2007....
; GÖTSCH et al., 2009Götsch S, Leschnik M, Duscher G, Burgstaller JP, Wille-Piazzai W, Joachim A. Ticks and haemoparasites of dogs from Praia, Cape Verde. Vet Parasitol 2009; 166(1-2): 171-174. PMid:19729247. http://dx.doi.org/10.1016/j.vetpar.2009.08.009
http://dx.doi.org/10.1016/j.vetpar.2009....
; DYACHENKO et al., 2012Dyachenko V, Pantchev N, Balzer HJ, Meyersen A, Straubinger RK. First case of Anaplasma platys infection in a dog from Croatia. Parasit Vectors 2012; 5: 49. http://dx.doi.org/10.1186/1756-3305-5-49
http://dx.doi.org/10.1186/1756-3305-5-49...
).

Anemia and thrombocytopenia are common findings associated withA. platys infection, even in asymptomatic dogs (HARVEY, 2006Harvey JW. Thrombocytitropic Anaplasmosis (A. platys [E. platys] Infection). In: Greene CE. Infectious Diseases of the Dog and Cat. 3rd ed. St. Louis: Elsevier; 2006. p. 229-231.; FERREIRA et al., 2008bFerreira RF, Cerqueira AMF, Pereira AM, Ferreira MS, Almosny NRP. Hematologic Parameters in Polymerase Chain Reaction-Positive and -Negative Dogs for Anaplasma platys Presenting Platelet Inclusion Bodies. Intern J Appl Res Vet Med 2008b; 6(3): 185-190.). However, occurrences of anemia or thrombocytopenia did not show any association with positive PCR or serological findings in the present study (Table 1). It can be explained by bad nutritional status, since most of these dogs are fed food scraps, garbage or low quality diet. These co-finding factors might have interfered with the statistical analyses.

The WBC varied among the dogs in the present study, which is consistent with previous reports of A. platys infection showing WBC within the reference range (AGUIRRE et al., 2006Aguirre E, Tesouro MA, Ruiz L, Amusategui I, Sainz A. Genetic Characterization of Anaplasma (Ehrlichia) platys in Dogs in Spain. J Vet Med B Infect Dis Vet Public Health 2006; 53(4): 197-200. PMid:16629989. http://dx.doi.org/10.1111/j.1439-0450.2006.00937.x
http://dx.doi.org/10.1111/j.1439-0450.20...
) or greater than this range (BEAUFILS et al., 2002Beaufils JP, Inokuma H, Martin-Granel J, Jumelle P, Barbault-Jumelle 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(2): 85-90.; ULUTAS et al., 2007Ulutas B, Bayramli G, Karagenç T. First Case of Anaplasma (Ehrlichia) platys Infection in a Dog in Turkey. Turk J Vet Anim Sci 2007; 31(4): 279-282.). The basophil count above the reference values for the leukocyte differential was the only significant difference (p = 0.015) associated with nPCR positive dogs. Although no previous study has reported basophilia in association with A. platys infection, basophils are known to chemotactically respond to bacterial products (RIZZI et al., 2010).

This was the first molecular study to survey the presence of the vector-borne pathogens E. canis and A. platys in domestic dogs in Porto Alegre, Southern Brazil. The present results indicate that CICT caused by A. platys may be endemic in this area. AlthoughA. platys is considered to be less pathogenic than other species of the Anaplasmataceae family, such as E. canis, the impact of A. platys infection on animal health should not be underestimated, since infection may increase the risk of other diseases (CARDOZO et al., 2009Cardozo GP, Oliveira LP, Mansur MAB, Santos EV, Roberto PG, Marins M. Molecular characterisation of two strains of Anaplasma platys in Brazil. Vet Rec 2009; 164(11): 338-339. PMid:19287032. http://dx.doi.org/10.1136/vr.164.11.338
http://dx.doi.org/10.1136/vr.164.11.338...
; GAUNT et al., 2010Gaunt SD, Beall MJ, Stillman BA, Lorentzen L, Diniz PPVP, Chandrashekar R, Breitschwerdt EB. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasites & Vectors 2010; 3:33. PMid:20377870 PMCid:PMC2859368. http://dx.doi.org/10.1186/1756-3305-3-33
http://dx.doi.org/10.1186/1756-3305-3-33...
). Our findings should be further investigated in order to fully establish the impact of canine cyclic thrombocytopenia in dogs and their potential reservoir role and co-infections withA. platys in Southern Brazil.

Conclusions

In this study, we reported on occurrences and molecular detection ofA. platys in naturally infected dogs in Southern Brazil for the first time. Our results showed that for an accurate diagnosis, serological and molecular methods should be combined, since there was no correlation between PCR and serological findings, and no hematological abnormalities were associated withA. platys infection.

Although dogs are commonly infected with E. canis in Brazil, all the dogs from the Zoonosis Control Center and from Arquipelago of Porto Alegre, Southern Brazil, tested in this study were negative for E. canis infection. Thus, the prevalence of E. canis in these areas is either low or absent. The ELISA test for these organisms has not previously been validated for strains of Anaplasma andEhrlichia in Brazil, and despite its widespread use in routine clinical analyses in Brazil, it may be flawed with regard to identifying native species.

This work was supported by the National Scientific and Technological Development Council (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq). The authors thank Dr. Ahmed Mohamed for performing the statistical analyses and IDEXX Laboratories for providing the SNAP 4Dx® tests.

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Publication Dates

  • Publication in this collection
    Jul-Sep 2013

History

  • Received
    26 Feb 2013
  • Accepted
    10 July 2013
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br