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Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA
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Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA Daniel M. Aguiar a,b,∗ , Xiaofeng Zhang a , Isis A. Braga b , Isis I.G.G. Taques b , Jere W. McBride a a Department of Pathology, Center of Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA b Laboratório de Virologia e Rickettsioses, Hospital Veterinário da Universidade Federal de Mato Grosso (HOVET-UFMT), Cuiabá, MT, Brazil
a r t i c l e
i n f o
Article history: Received 28 July 2015 Received in revised form 2 October 2015 Accepted 9 October 2015 Available online xxx Keywords: Ehrlichia Tandem repeat protein ELISA Genotype Immunodiagnosis Brazil
a b s t r a c t We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n = 9) or Br TRP36 (n = 6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n = 28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Ehrlichia canis is the etiologic agent of the globally distributed tick-borne disease, canine monocytic ehrlichiosis (CME), a chronic and sometimes lethal disease of dogs (Zweygarth et al., 2014). Despite the worldwide distribution of E. canis, 16S rRNA gene sequences are 99.4–100% identical among isolates from dispersed countries and provide little information regarding the overall diversity of this organism. Immunoreactive proteins including the OMP-1 family, thiodisulfide oxidoreductase (Dsb), and the tandem repeat proteins (TRP) 19 and 140 have also been found to be conserved in geographically dispersed strains (Zhang et al., 2008; Yu et al., 2007; Aguiar et al., 2008; Kamani et al., 2013). However, differences in the TRP36 gene have been reported, indicating substantial degree of E. canis diversity (Doyle et al., 2005; Zhang
∗ Corresponding author at: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA. E-mail address: [email protected] (D.M. Aguiar).
et al., 2008; Hsieh et al., 2010; Aguiar et al., 2013; Zweygarth et al., 2014). The TRP19 and TRP36 of E. canis are major immunoreactive proteins and primary targets of host antibody response during infection. The sensitivities and specificities of these proteins for species-specific immunodiagnosis have been determined using recombinant proteins and synthetic peptide based assays (Doyle et al., 2006; Cárdenas et al., 2007; McBride et al., 2007). The TRP19 is highly conserved among the known E. canis strains (McBride et al., 2007; Zhang et al., 2008). Conversely, variability in the TRP36 gene among isolates from the US, Israel, Taiwan, and Brazil has indicated that this gene can be utilized to define E. canis strain diversity since a variable amino acid tandem repeat sequences have been reported. For example, the TR amino acid sequence of the US TRP36 has been detected on different continents (Doyle et al., 2005; Hsieh et al., 2010; Kamani et al., 2013; Zweygarth et al., 2014), while the different TR amino acid sequences have been reported in Israel and Brazil (Zhang et al., 2008; Aguiar et al., 2013). Major antibody epitopes have been characterized in the tandem repeat regions of TRPs from E. canis and E. chaffeensis, and these
http://dx.doi.org/10.1016/j.ttbdis.2015.10.003 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003
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epitopes are molecularly distinct enabling detection of speciesspecific antibodies that allow serologic differentiation of the infecting agent (Doyle et al., 2006; McBride et al., 2007; Luo et al., 2008). In this study, we developed an ELISA-based peptide assay to detect and distinguish E. canis infections by two defined TRP36 genotypes in Brazil, and provide serologic evidence of genotypespecific infections as well as coinfections or multiple infections with both TRP36 genotypes. 2. Materials and methods 2.1. Dog sera Sera from 30 dogs naturally infected with E. canis were obtained during serological surveys in the Brazilian States of Rondonia, Pará, Paraná, São Paulo and Mato Grosso, which antibody status to E. canis was previously determined by IFA (titers ranging from 160 to 327,680) (Aguiar et al., 2007; Melo et al., 2011). E. canis was previously isolated and molecularly characterized from six of these dogs (MN#24, Cba#1, Cba#16, Ld#1, PP#14 and BL#6) (Aguiar et al., 2013). Positive control sera from dogs experimentally infected with E. canis US TRP36 genotype (Jake strain), E. canis Brazilian TRP36 genotype (Br TRP36, Cba #1 strain) and E. chaffeensis (Arkansas strain) (dogs #2995, #B2015 and #2251 respectively) and sera from 12 dogs from the US and Brazil that did not have E. canis antibodies determined by IFA were also included in the experiment as positive and negative controls. 2.2. Synthetic peptides and ELISA Peptides corresponding to the repeat regions of TRP36 from US E. canis strain (18-mer, TEDSVSAPATEDSVSAPA) (Doyle et al., 2006), the Brazilian E. canis strain (24-mer, ASVVPEAEASVVPEAEASVVPEAE) (Aguiar et al., 2013) and the TRP19 E. canis epitope-containing region (24-mer, HFTGPTFSEVNLSEEEKMELQEVS) (McBride et al., 2007) were synthetized (Bio-Synthesis Inc., Lewisville, TX). A peptide corresponding to the TRP36 C-terminal region from the E. canis Israeli strain (IS36-C-V, 15-mer, NPTGLKFLDLYTQLTL) (Zhang et al., 2008) was used as a negative peptide control. All peptides (lyophilized) were resuspended in molecular grade water at 1 mg/mL. ELISA plates (MaxiSorp; Nunc, Roskilde, Denamark) were coated (1.0 g/well) with respective synthetic peptides suspended in phosphate-buffered saline (pH 7.4) and the assay was performed as previously described (Luo et al., 2010). The color development was determined on a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA), and data were analyzed by using SoftMax Pro v4.0 (Molecular Devices). Optical density (OD; A650 ) readings represent the mean for three wells (±standard deviations) after subtracting the OD value of the non-reactive control peptide (IS36-C-V). All sera IFA negative for E. canis and the E. chaffeensis-infected dog had mean OD readings of <0.020 and <0.100, respectively; therefore a positive sample threshold was set at >0.300 OD units above the negative control absorbance. Specificity value for the ELISA was calculated according to Thrusfield (2007) and the confidence interval of 95% were calculated for the results of serum samples of naturally infected dogs by the EpiInfo 7.0 software for windows. 3. Results All Brazilian dog samples had antibodies that reacted against at least one E. canis peptide. The OD values of naturally E. canis infected dogs are shown in Table 1. Nineteen (63.3%, 95% IC: 43.8–80.0%) sera had antibodies specific for the Br TRP36 peptide, 22 (73.3%, 95% IC: 54.1–87.7%) dogs had antibody that reacted with US TRP36 peptide,
Table 1 Reactivity of synthetic peptides from TRP36 (Brazilian and US genotype) and TRP19 with antibodies in sera of dogs naturally infected with E. canis by ELISA. Dog
#22 #37 42C1 45C1 49C1 61C4 82C2 #93 #107 U#4 U#8 U#37 U#45 U#72 R#49 R#55 R#57 R#62 R#69 Dani SPT BL#1 BL#3 BL#9 MN#24 Cba #1 Cba#16 LD#1 PP#14 BL#6
IFA titers
20,480 20,480 20,480 20,480 20,480 1280 1280 5120 10,240 2560 2560 2560 160 20,480 327,680 327,680 40,960 81,920 1280 10,240 2560 40,690 640 163,840 81,920 81,920 10,240 10,240 640 2560
Optical density (OD; A650 )a TRP19
Br TRP36
US TRP36
0.798 2.040 0.571 0.544 0.222 0.039 0.542 0.332 0.598 2.509 3.015 2.139 1.828 3.258 2.861 2.689 2.721 0.840 0.579 1.870 1.876 0.913 0.300 1.365 2.269 2.660 0.385 2.702 2.869 2.671
0.144 0.138 0.191 0.002 0.382 0.080 0.070 0.116 1.837 0.653 0.241 0.161 0.364 2.864 0.347 2.261 1.876 1.656 0.111 1.739 0.003 3.082 1.112 2.183 1.955 2.326 0.369 0.311 3.698 2.007
2.295 2.712 1.818 2.576 0.020 0.828 0.079 0.781 0,142 1.964 1.580 2.849 1.793 2.752 1.768 2.060 2.263 0.169 0.038 2.829 0.407 1.748 0.248 2.011 2.081 1.049 2.499 0.160 3.202 0.009
a The OD readings represent the means for three wells with the OD of the control (IS36C-V) wells subtracted.
and 28 (93.3%, 95% IC: 78.0–99.2%) dogs had antibody to the TRP19 peptide. Six (20%) dogs had antibodies to the Br TRP36 peptide, but not the US TRP36, and 9 (30%) dogs (30%) had antibodies that reacted with the US TRP36 peptide, but not the Br TRP36 peptide. Antibodies that reacted with both TRP36 peptides were detected in 13 (43.3%) samples. One dog (61C4) had antibodies specific only for US TRP36 peptide, and the dog 49C1 had antibodies specific only for Br TRP36 peptide. The dogs 82C2 and R69 had antibodies against the TRP19 peptide, but not for either Br or US TRP36 peptides. The mean OD value of Br TRP36 peptides of positive dogs was 1.630, US TRP36 positive dogs was 1.994 and TRP19 positive dogs was 1.705. The mean OD value of IS36-C peptides was 0.101. The results of dogs naturally infected with E. canis in Brazil were shown in Fig. 1. The synthetic peptides exhibited 100% specificity in the ELISA (Fig. 2). The results were compared to the results of IFA with sera from healthy (sera negative) dogs and US TRP36 E. canis (#2995), Br TRP36 E. canis (#2015) and E. chaffeensis (#2551) experimentally infected dogs. None of the IFA-negative dogs reacted positively with synthetic peptides. The serum from the US TRP36 E. canis-infected dog had a titer of 10,240 by IFA, and reacted against US TRP36 peptide with mean OD of 0.963 and against TRP19 with mean OD of 1.681. The mean values for Br TRP36 and IS36-C-V were 0.171 and 0.097 respectively. The serum of Br TRP36 E. canis infected dog had a titer of 20,480 by IFA and reacted against Br TRP36 peptide with mean OD of 2.113 and against TRP19 with mean OD of 2.097. The mean values for US TRP36 and IS36-C-V were 0.055 and 0.063 respectively. The sera of E. chaffeensis infected dogs had a titer of 10,240 by IFA (DH82 cells infected with E. chaffeensis) and showed mean OD values of 0.091 for Br TRP36, 0.077 for US TRP36, 0.091 for TRP19 and 0.069 for IS36-C-V peptides. All negative control sera (IFA < 64) were negative for all peptides and the mean ELISA OD value was 0.053.
Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003
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Fig. 1. Reactivity of synthetic peptides derived from TRP36 (Brazilian and US genotype) and TRP19 with antibody in sera of dogs by ELISA, which different E. canis genotypes were isolated and characterized according to Aguiar et al. (2013). The OD readings represent the means for three wells with the OD of the negative control (IS36C-V) wells subtracted. The cutoff OD (0.3) established denoted by a dotted line indicates the threshold for a positive reading.
Fig. 2. Reactivity of synthetic peptides from TRP36 (Brazilian and US genotype) and TRP19 with antibodies in sera of healthy dogs and sera of dogs experimentally infected with the US and Br genotypes of E. canis (#2995 and #B2015) and E. chaffeensis (#2551). The OD readings represent the means for three wells with the OD of the control (IS36C-V) wells subtracted. The cutoff OD (0.3) established denoted by a dotted line indicates the threshold for a positive reading.
4. Discussion We have previously reported a novel E. canis genotype in Brazil (Aguiar et al., 2013), and there are likely other novel E. canis genotypes circulating in Brazil that can be molecularly distinguished by the TRP36. Serologic assays to understand E. canis infections by each genotype will be useful for diagnosis, understanding clinical manifestations represented by each strain, and for understanding the epidemiology of canine ehrlichiosis in Brazil. ELISA using synthetic peptides to serologically distinguish E. canis and E. chaffeensis infections have been previously reported (Doyle et al., 2006; McBride et al., 2007; Luo et al., 2008). Thus, based on the molecular identification and characterization of the E. canis TRP36 genotypes in Brazil (Aguiar et al., 2013), we used this molecular divergence in TRP36 genes to develop an ELISA capable of serologically distinguishing antibodies against two different E. canis genotypes using synthetic peptides that represent the major epitope in TRP36. The ELISA based on Br and US TRP36 peptide demonstrated high specificity since nonspecific antibody reactivity was not observed with sera of IFA negative dogs. Moreover, antibodies in sera from dogs experimentally infected with the US TRP36 and Br TRP36
genotypes of E. canis and E. chaffeensis Arkansas strain did not crossreact between each other, indicating that both US and Br TRP36 peptide is molecularly distinct and reacts only with antibodies generated against homologous genotype. A similar result was observed when different reactivity was found in sera of E. chaffeensis human patients tested by an ELISA developed with the ortholog TRP47 of E. chaffeensis synthetic peptides (Luo et al., 2010). Distinct TRP47 genotypes have also been described in the Arkansas and Sapulpa strains of E. chaffeensis that did not serologically cross react (Doyle et al., 2006). Most dogs had antibodies that reacted with the TRP19 peptide, which is a highly conserved immunoreactive protein of E. canis (McBride et al., 2007). Moreover, one dog that had antibodies to the TRP19 peptide did not react with either TRP36 peptide, suggesting that this dog may have been exposed to a novel E. canis TRP36 genotype circulating in Brazil. On the other hand, the occurrence of specific antibodies against both genotypes in the same dog implicate that dogs can be co-infected or infected at different times by different genotypes. This result supports the possibility of genetic recombination occurring between the US and Br TRP36 genotypes, which may have occurred in a previous study in which
Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003
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a strain of E. canis (Cba#16) showed an amino acid sequence in the TR region identical to the US genotype but a N-terminal amino acid sequence most similar to the Brazilian genotype (Aguiar et al., 2013). In addition, serum samples of dogs Cba#1 and MN#24 collected in different regions from Brazil, which the Brazilian genotype were isolated previously showed reactivity for US and Br TRP36 peptides indicating that both US and Br strains were circulating in their regions. Finally, a single positivity for the US TRP36 and Br TRP36 peptide observed in dogs 61C4 and 49C1 highlights the applicability of TRP36 as a sensitive antigen to be used in the immunodiagnostic test of CME. Moreover, the synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 443923/2014-0). We thank Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) for the pos-doctoral scholarship program granted to D.M. Aguiar and for the graduate scholarships of I.A. Braga and I.I.G.G. Taques. A special thanks to F.G. Aguiar, A.C. Gaíva and G.G. Aguiar for their unwavering support during the sabbatical in which this study was completed. References Aguiar, D.M., Cavalcante, G.T., Pinter, A., Gennari, S.M., Camargo, L.M., Labruna, M.B., 2007. Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil. J. Med. Entomol. 44, 126–132. Aguiar, D.M., Hagiwara, M.K., Labruna, M.B., 2008. In vitro isolation and molecular characterization of an Ehrlichia canis strain from São Paulo, Brazil. Braz. J. Microbiol. 39, 489–493. Aguiar, D.M., Zhang, X., Melo, A.L., Pacheco, T.A., Meneses, A.M., Zanutto, M.S., Horta, M.C., Santarém, V.A., Camargo, L.M., McBride, J.W., Labruna, M.B., 2013. Genetic diversity of Ehrlichia canis in Brazil. Vet. Microbiol. 164, 315–321.
Cárdenas, A.M., Doyle, C.K., Zhang, X., Nethery, K., Corstvet, R.E., Walker, D.H., McBride, J.W., 2007. Enzyme-linked immunosorbent assay with conserved immunoreactive glycoproteins gp36 and gp19 has enhanced sensitivity and provides species-specific immunodiagnosis of Ehrlichia canis infection. Clin. Vaccine Immunol. 14, 123–128. Doyle, C.K., Cardenas, A.M., Aguiar, D.M., Labruna, M.B., Ndip, L.M., Yu, X.J., McBride, J.W., 2005. Molecular characterization of E. canis gp36 and E. chaffeensis gp47 tandem repeats among isolates from different geographic locations. Ann. N. Y. Acad. Sci. 1063, 433–435. Doyle, C.K., Nethery, K.A., Popov, V.L., McBride, J.W., 2006. Differentially expressed and secreted major immunoreactive protein orthologs of Ehrlichia canis and E. chaffeensis elicit early antibody responses to epitopes on glycosylated tandem repeats. Infect. Immun. 74, 711–720. Hsieh, Y.C., Lee, C.C., Tsang, C.L., Chung, Y.T., 2010. Detection and characterization of four novel genotypes of Ehrlichia canis from dogs. Vet. Microbiol. 146, 70–75. Kamani, J., Lee, C.C., Haruna, A.M., Chung, P.J., Weka, P.R., Chung, Y.T., 2013. First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria. Res. Vet. Sci. 94, 27–32. Luo, T., Zhang, X., Wakeel, A., Popov, V.L., McBride, J.W., 2008. A variable-length PCR target protein of Ehrlichia chaffeensis contains major species-specific antibody epitopes in acidic serine-rich tandem repeats. Infect. Immun. 76, 1572–1580. Luo, T., Zhang, X., Nicholson, W.L., Zhu, B., McBride, J.W., 2010. Molecular characterization of antibody epitopes of Ehrlichia chaffeensis ankyrin protein 200 and tandem repeat protein 47 and evaluation of synthetic immunodeterminants for serodiagnosis of human monocytotropic ehrlichiosis. Clin. Vaccine Immunol. 17, 87–97. McBride, J.W., Doyle, C.K., Zhang, X., Cardenas, A.M., Popov, V.L., Nethery, K.A., Woods, M.E., 2007. Identification of a glycosylated Ehrlichia canis 19-kilodalton major immunoreactive protein with a species-specific serine-rich glycopeptide epitope. Infect. Immun. 75, 74–82. Melo, A.L.T., Martins, T.F., Horta, M.C., Moraes-Filho, J., Pacheco, R.C., Labruna, M.B., Aguiar, D.M., 2011. Seroprevalence and risk factors to Ehrlichia spp. and Rickettsia spp. in dogs from the Pantanal Region of Mato Grosso State, Brazil. Ticks Tick Borne Dis. 2, 213–218. Thrusfield, M., 2007. The nature of data. In: Thrusfield, M. (Ed.), Veterinary Epidemiology. Wiley-Blackwell, New Jersey, pp. 152–166. Yu, X.Z., McBride, J.W., Walker, D.H., 2007. Restriction and expansion of Ehrlichia strain diversity. Vet. Microbiol. 143, 337–346. Zhang, X., Luo, T., Keysary, A., Baneth, G., Miyashiro, S., Strenger, C., Waner, T., McBride, J.W., 2008. Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains. Clin. Vaccine Immunol. 15, 1080–1088. Zweygarth, E., Cabezas-Cruz, A., Josemans, A.I., Oosthuizen, M.C., Matjila, P.T., Lis, K., Broniszewska, M., Schöl, H., Ferrolho, J., Grubhoffer, L., Passos, L.M., 2014. In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates. Ticks Tick Borne Dis. 5, 423–431.
Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA Daniel M. Aguiar a,b,∗ , Xiaofeng Zhang a , Isis A. Braga b , Isis I.G.G. Taques b , Jere W. McBride a a Department of Pathology, Center of Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA b Laboratório de Virologia e Rickettsioses, Hospital Veterinário da Universidade Federal de Mato Grosso (HOVET-UFMT), Cuiabá, MT, Brazil
a r t i c l e
i n f o
Article history: Received 28 July 2015 Received in revised form 2 October 2015 Accepted 9 October 2015 Available online xxx Keywords: Ehrlichia Tandem repeat protein ELISA Genotype Immunodiagnosis Brazil
a b s t r a c t We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n = 9) or Br TRP36 (n = 6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n = 28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Ehrlichia canis is the etiologic agent of the globally distributed tick-borne disease, canine monocytic ehrlichiosis (CME), a chronic and sometimes lethal disease of dogs (Zweygarth et al., 2014). Despite the worldwide distribution of E. canis, 16S rRNA gene sequences are 99.4–100% identical among isolates from dispersed countries and provide little information regarding the overall diversity of this organism. Immunoreactive proteins including the OMP-1 family, thiodisulfide oxidoreductase (Dsb), and the tandem repeat proteins (TRP) 19 and 140 have also been found to be conserved in geographically dispersed strains (Zhang et al., 2008; Yu et al., 2007; Aguiar et al., 2008; Kamani et al., 2013). However, differences in the TRP36 gene have been reported, indicating substantial degree of E. canis diversity (Doyle et al., 2005; Zhang
∗ Corresponding author at: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA. E-mail address: [email protected] (D.M. Aguiar).
et al., 2008; Hsieh et al., 2010; Aguiar et al., 2013; Zweygarth et al., 2014). The TRP19 and TRP36 of E. canis are major immunoreactive proteins and primary targets of host antibody response during infection. The sensitivities and specificities of these proteins for species-specific immunodiagnosis have been determined using recombinant proteins and synthetic peptide based assays (Doyle et al., 2006; Cárdenas et al., 2007; McBride et al., 2007). The TRP19 is highly conserved among the known E. canis strains (McBride et al., 2007; Zhang et al., 2008). Conversely, variability in the TRP36 gene among isolates from the US, Israel, Taiwan, and Brazil has indicated that this gene can be utilized to define E. canis strain diversity since a variable amino acid tandem repeat sequences have been reported. For example, the TR amino acid sequence of the US TRP36 has been detected on different continents (Doyle et al., 2005; Hsieh et al., 2010; Kamani et al., 2013; Zweygarth et al., 2014), while the different TR amino acid sequences have been reported in Israel and Brazil (Zhang et al., 2008; Aguiar et al., 2013). Major antibody epitopes have been characterized in the tandem repeat regions of TRPs from E. canis and E. chaffeensis, and these
http://dx.doi.org/10.1016/j.ttbdis.2015.10.003 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003
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epitopes are molecularly distinct enabling detection of speciesspecific antibodies that allow serologic differentiation of the infecting agent (Doyle et al., 2006; McBride et al., 2007; Luo et al., 2008). In this study, we developed an ELISA-based peptide assay to detect and distinguish E. canis infections by two defined TRP36 genotypes in Brazil, and provide serologic evidence of genotypespecific infections as well as coinfections or multiple infections with both TRP36 genotypes. 2. Materials and methods 2.1. Dog sera Sera from 30 dogs naturally infected with E. canis were obtained during serological surveys in the Brazilian States of Rondonia, Pará, Paraná, São Paulo and Mato Grosso, which antibody status to E. canis was previously determined by IFA (titers ranging from 160 to 327,680) (Aguiar et al., 2007; Melo et al., 2011). E. canis was previously isolated and molecularly characterized from six of these dogs (MN#24, Cba#1, Cba#16, Ld#1, PP#14 and BL#6) (Aguiar et al., 2013). Positive control sera from dogs experimentally infected with E. canis US TRP36 genotype (Jake strain), E. canis Brazilian TRP36 genotype (Br TRP36, Cba #1 strain) and E. chaffeensis (Arkansas strain) (dogs #2995, #B2015 and #2251 respectively) and sera from 12 dogs from the US and Brazil that did not have E. canis antibodies determined by IFA were also included in the experiment as positive and negative controls. 2.2. Synthetic peptides and ELISA Peptides corresponding to the repeat regions of TRP36 from US E. canis strain (18-mer, TEDSVSAPATEDSVSAPA) (Doyle et al., 2006), the Brazilian E. canis strain (24-mer, ASVVPEAEASVVPEAEASVVPEAE) (Aguiar et al., 2013) and the TRP19 E. canis epitope-containing region (24-mer, HFTGPTFSEVNLSEEEKMELQEVS) (McBride et al., 2007) were synthetized (Bio-Synthesis Inc., Lewisville, TX). A peptide corresponding to the TRP36 C-terminal region from the E. canis Israeli strain (IS36-C-V, 15-mer, NPTGLKFLDLYTQLTL) (Zhang et al., 2008) was used as a negative peptide control. All peptides (lyophilized) were resuspended in molecular grade water at 1 mg/mL. ELISA plates (MaxiSorp; Nunc, Roskilde, Denamark) were coated (1.0 g/well) with respective synthetic peptides suspended in phosphate-buffered saline (pH 7.4) and the assay was performed as previously described (Luo et al., 2010). The color development was determined on a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA), and data were analyzed by using SoftMax Pro v4.0 (Molecular Devices). Optical density (OD; A650 ) readings represent the mean for three wells (±standard deviations) after subtracting the OD value of the non-reactive control peptide (IS36-C-V). All sera IFA negative for E. canis and the E. chaffeensis-infected dog had mean OD readings of <0.020 and <0.100, respectively; therefore a positive sample threshold was set at >0.300 OD units above the negative control absorbance. Specificity value for the ELISA was calculated according to Thrusfield (2007) and the confidence interval of 95% were calculated for the results of serum samples of naturally infected dogs by the EpiInfo 7.0 software for windows. 3. Results All Brazilian dog samples had antibodies that reacted against at least one E. canis peptide. The OD values of naturally E. canis infected dogs are shown in Table 1. Nineteen (63.3%, 95% IC: 43.8–80.0%) sera had antibodies specific for the Br TRP36 peptide, 22 (73.3%, 95% IC: 54.1–87.7%) dogs had antibody that reacted with US TRP36 peptide,
Table 1 Reactivity of synthetic peptides from TRP36 (Brazilian and US genotype) and TRP19 with antibodies in sera of dogs naturally infected with E. canis by ELISA. Dog
#22 #37 42C1 45C1 49C1 61C4 82C2 #93 #107 U#4 U#8 U#37 U#45 U#72 R#49 R#55 R#57 R#62 R#69 Dani SPT BL#1 BL#3 BL#9 MN#24 Cba #1 Cba#16 LD#1 PP#14 BL#6
IFA titers
20,480 20,480 20,480 20,480 20,480 1280 1280 5120 10,240 2560 2560 2560 160 20,480 327,680 327,680 40,960 81,920 1280 10,240 2560 40,690 640 163,840 81,920 81,920 10,240 10,240 640 2560
Optical density (OD; A650 )a TRP19
Br TRP36
US TRP36
0.798 2.040 0.571 0.544 0.222 0.039 0.542 0.332 0.598 2.509 3.015 2.139 1.828 3.258 2.861 2.689 2.721 0.840 0.579 1.870 1.876 0.913 0.300 1.365 2.269 2.660 0.385 2.702 2.869 2.671
0.144 0.138 0.191 0.002 0.382 0.080 0.070 0.116 1.837 0.653 0.241 0.161 0.364 2.864 0.347 2.261 1.876 1.656 0.111 1.739 0.003 3.082 1.112 2.183 1.955 2.326 0.369 0.311 3.698 2.007
2.295 2.712 1.818 2.576 0.020 0.828 0.079 0.781 0,142 1.964 1.580 2.849 1.793 2.752 1.768 2.060 2.263 0.169 0.038 2.829 0.407 1.748 0.248 2.011 2.081 1.049 2.499 0.160 3.202 0.009
a The OD readings represent the means for three wells with the OD of the control (IS36C-V) wells subtracted.
and 28 (93.3%, 95% IC: 78.0–99.2%) dogs had antibody to the TRP19 peptide. Six (20%) dogs had antibodies to the Br TRP36 peptide, but not the US TRP36, and 9 (30%) dogs (30%) had antibodies that reacted with the US TRP36 peptide, but not the Br TRP36 peptide. Antibodies that reacted with both TRP36 peptides were detected in 13 (43.3%) samples. One dog (61C4) had antibodies specific only for US TRP36 peptide, and the dog 49C1 had antibodies specific only for Br TRP36 peptide. The dogs 82C2 and R69 had antibodies against the TRP19 peptide, but not for either Br or US TRP36 peptides. The mean OD value of Br TRP36 peptides of positive dogs was 1.630, US TRP36 positive dogs was 1.994 and TRP19 positive dogs was 1.705. The mean OD value of IS36-C peptides was 0.101. The results of dogs naturally infected with E. canis in Brazil were shown in Fig. 1. The synthetic peptides exhibited 100% specificity in the ELISA (Fig. 2). The results were compared to the results of IFA with sera from healthy (sera negative) dogs and US TRP36 E. canis (#2995), Br TRP36 E. canis (#2015) and E. chaffeensis (#2551) experimentally infected dogs. None of the IFA-negative dogs reacted positively with synthetic peptides. The serum from the US TRP36 E. canis-infected dog had a titer of 10,240 by IFA, and reacted against US TRP36 peptide with mean OD of 0.963 and against TRP19 with mean OD of 1.681. The mean values for Br TRP36 and IS36-C-V were 0.171 and 0.097 respectively. The serum of Br TRP36 E. canis infected dog had a titer of 20,480 by IFA and reacted against Br TRP36 peptide with mean OD of 2.113 and against TRP19 with mean OD of 2.097. The mean values for US TRP36 and IS36-C-V were 0.055 and 0.063 respectively. The sera of E. chaffeensis infected dogs had a titer of 10,240 by IFA (DH82 cells infected with E. chaffeensis) and showed mean OD values of 0.091 for Br TRP36, 0.077 for US TRP36, 0.091 for TRP19 and 0.069 for IS36-C-V peptides. All negative control sera (IFA < 64) were negative for all peptides and the mean ELISA OD value was 0.053.
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Fig. 1. Reactivity of synthetic peptides derived from TRP36 (Brazilian and US genotype) and TRP19 with antibody in sera of dogs by ELISA, which different E. canis genotypes were isolated and characterized according to Aguiar et al. (2013). The OD readings represent the means for three wells with the OD of the negative control (IS36C-V) wells subtracted. The cutoff OD (0.3) established denoted by a dotted line indicates the threshold for a positive reading.
Fig. 2. Reactivity of synthetic peptides from TRP36 (Brazilian and US genotype) and TRP19 with antibodies in sera of healthy dogs and sera of dogs experimentally infected with the US and Br genotypes of E. canis (#2995 and #B2015) and E. chaffeensis (#2551). The OD readings represent the means for three wells with the OD of the control (IS36C-V) wells subtracted. The cutoff OD (0.3) established denoted by a dotted line indicates the threshold for a positive reading.
4. Discussion We have previously reported a novel E. canis genotype in Brazil (Aguiar et al., 2013), and there are likely other novel E. canis genotypes circulating in Brazil that can be molecularly distinguished by the TRP36. Serologic assays to understand E. canis infections by each genotype will be useful for diagnosis, understanding clinical manifestations represented by each strain, and for understanding the epidemiology of canine ehrlichiosis in Brazil. ELISA using synthetic peptides to serologically distinguish E. canis and E. chaffeensis infections have been previously reported (Doyle et al., 2006; McBride et al., 2007; Luo et al., 2008). Thus, based on the molecular identification and characterization of the E. canis TRP36 genotypes in Brazil (Aguiar et al., 2013), we used this molecular divergence in TRP36 genes to develop an ELISA capable of serologically distinguishing antibodies against two different E. canis genotypes using synthetic peptides that represent the major epitope in TRP36. The ELISA based on Br and US TRP36 peptide demonstrated high specificity since nonspecific antibody reactivity was not observed with sera of IFA negative dogs. Moreover, antibodies in sera from dogs experimentally infected with the US TRP36 and Br TRP36
genotypes of E. canis and E. chaffeensis Arkansas strain did not crossreact between each other, indicating that both US and Br TRP36 peptide is molecularly distinct and reacts only with antibodies generated against homologous genotype. A similar result was observed when different reactivity was found in sera of E. chaffeensis human patients tested by an ELISA developed with the ortholog TRP47 of E. chaffeensis synthetic peptides (Luo et al., 2010). Distinct TRP47 genotypes have also been described in the Arkansas and Sapulpa strains of E. chaffeensis that did not serologically cross react (Doyle et al., 2006). Most dogs had antibodies that reacted with the TRP19 peptide, which is a highly conserved immunoreactive protein of E. canis (McBride et al., 2007). Moreover, one dog that had antibodies to the TRP19 peptide did not react with either TRP36 peptide, suggesting that this dog may have been exposed to a novel E. canis TRP36 genotype circulating in Brazil. On the other hand, the occurrence of specific antibodies against both genotypes in the same dog implicate that dogs can be co-infected or infected at different times by different genotypes. This result supports the possibility of genetic recombination occurring between the US and Br TRP36 genotypes, which may have occurred in a previous study in which
Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003
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a strain of E. canis (Cba#16) showed an amino acid sequence in the TR region identical to the US genotype but a N-terminal amino acid sequence most similar to the Brazilian genotype (Aguiar et al., 2013). In addition, serum samples of dogs Cba#1 and MN#24 collected in different regions from Brazil, which the Brazilian genotype were isolated previously showed reactivity for US and Br TRP36 peptides indicating that both US and Br strains were circulating in their regions. Finally, a single positivity for the US TRP36 and Br TRP36 peptide observed in dogs 61C4 and 49C1 highlights the applicability of TRP36 as a sensitive antigen to be used in the immunodiagnostic test of CME. Moreover, the synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 443923/2014-0). We thank Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) for the pos-doctoral scholarship program granted to D.M. Aguiar and for the graduate scholarships of I.A. Braga and I.I.G.G. Taques. A special thanks to F.G. Aguiar, A.C. Gaíva and G.G. Aguiar for their unwavering support during the sabbatical in which this study was completed. References Aguiar, D.M., Cavalcante, G.T., Pinter, A., Gennari, S.M., Camargo, L.M., Labruna, M.B., 2007. Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil. J. Med. Entomol. 44, 126–132. Aguiar, D.M., Hagiwara, M.K., Labruna, M.B., 2008. In vitro isolation and molecular characterization of an Ehrlichia canis strain from São Paulo, Brazil. Braz. J. Microbiol. 39, 489–493. Aguiar, D.M., Zhang, X., Melo, A.L., Pacheco, T.A., Meneses, A.M., Zanutto, M.S., Horta, M.C., Santarém, V.A., Camargo, L.M., McBride, J.W., Labruna, M.B., 2013. Genetic diversity of Ehrlichia canis in Brazil. Vet. Microbiol. 164, 315–321.
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Please cite this article in press as: Aguiar, D.M., et al., Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.10.003