Veterinary Parasitology 205 (2014) 343–346
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Evaluation of 18S rDNA PCR assay using skin fragments as a diagnostic test for Trypanosoma caninum A.G.S. Pinto a,b , H.K. Toma c , F.B. Figueiredo d , M.F. Madeira b,∗ a Programa de pós-graduac¸ão em Pesquisa Clínica em Doenc¸as Infecciosas, Instituto de Pesquisa Clínica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil b Laboratório de Vigilância em Leishmanioses, Instituto de Pesquisa Clínica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil c Laboratório de Diagnóstico Molecular e Hematologia, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Rio de Janeiro, RJ, Brazil d Laboratório de Pesquisa Clínica em Dermatozoonoses de Animais Domésticos, Instituto de Pesquisa Clínica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 7 February 2014 Received in revised form 23 July 2014 Accepted 26 July 2014 Keywords: Trypanosoma caninum PCR Dog Leishmaniasis
a b s t r a c t Trypanosoma caninum is a new species that has been recently identified in Brazil and infects domestic dogs. To date, no accurate diagnostic assays for this parasite have been established; thus, our aim was to evaluate more than one type of PCR for the diagnosis and molecular screening of T. caninum in 229 dogs living in Rio de Janeiro state. The tests were based on the amplification and sequencing of the 18S ribosomal DNA (rDNA) gene using healthy skin fragments. Additionally, PCR amplification of the kDNA minicircles region specific to the Leishmania genus was performed. The PCR results were compared with those of culture-based analysis performed with the same specimen. Using cultures, T. caninum and Leishmania chagasi were isolated from 11 and 12 dogs, respectively, whereas the 18S rDNA PCR assay detected parasitic infection in 35 dogs. Among these, 25 dogs showed an amplification pattern similar to T. caninum and 10 showed a pattern similar to L. chagasi; these results were confirmed by sequencing analysis. The kDNA PCR analysis showed that 14 dogs were positive for Leishmania infection. Of these, 2 dogs showed negative culture results and 12 were positive for L. chagasi, including 4 with negative 18S rDNA PCR results. Thus far, culture-based testing has been the only tool used successfully for T. caninum diagnosis. Our results demonstrate that 18S rDNA PCR-based test should be a useful diagnostic tool, particularly for distinguishing between T. caninum and L. chagasi infections in areas where these 2 parasites co-exist. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma caninum was initially identified in a dog co-infected with Leishmania braziliensis in the municipality of Rio de Janeiro, and was subsequently detected in other
∗ Corresponding author. Tel.: +55 21 3865 9541; fax: +55 21 38659541. E-mail address: fatima.madeira@ipec.fiocruz.br (M.F. Madeira). http://dx.doi.org/10.1016/j.vetpar.2014.07.028 0304-4017/© 2014 Elsevier B.V. All rights reserved.
regions of Brazil (Madeira et al., 2009; Barros et al., 2012). Despite attempts to isolate this parasite from blood and tissue samples, all 53 isolates of T. caninum described to date have been obtained from cultures of healthy skin samples from dogs (Barros et al., 2012). This strongly suggests that skin specimens could be used for other assays, such as PCR-based tests. Although certain epidemiological aspects are still unknown, the presence of T. caninum in regions where
344
A.G.S. Pinto et al. / Veterinary Parasitology 205 (2014) 343–346
canine visceral leishmaniasis (CVL) is endemic has been regarded as a possible confounding factor in the control of leishmaniasis (Alves et al., 2012). Previous studies have reported that dogs that were infected with T. caninum and were also seropositive for Leishmania were euthanized, in agreement with the CVL control measures established in Brazil; however, the presence of Leishmania chagasi (syn. Leishmania infantum) was not confirmed in those animals (Pinto et al., 2010; Silva et al., 2011). These findings highlight the importance of using accurate methods that can clearly distinguish Leishmania spp. infections from T. caninum infections, especially in areas where both parasites co-exist. T. caninum is found in various CVL endemic regions in Brazil, but data regarding its prevalence rates are limited, particularly in the municipality of Rio de Janeiro where most cases have been registered (Barros et al., 2012). However, culture-based testing has been the only tool used successfully for T. caninum diagnosis to date. Therefore, our study aimed to assess a PCR-based tool targeting 18S ribosomal DNA (rDNA) for the diagnosis and screening of T. caninum infection in domestic dogs living in the municipality of Rio de Janeiro, Brazil.
18S rDNA gene (18S rDNA PCR) and involved a nested assay according to a previously described protocol (Smith et al., 2008; Barros et al., 2012). The amplification products were purified using a QIAquick Purification Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The nucleotide sequences were processed using an automated sequencer (3730 DNA Analyzer; Applied Biosystems), edited by BioEdit software, and analyzed using the Basic Local Alignment Search Tool (BLAST). The second PCR assay, performed for all samples with positive culture and/or 18S rDNA PCR test results, targeted a conserved region of kDNA minicircles specific to the Leishmania genus, based on protocols described by Degrave et al. (1994). The third assay targeted a constitutive canine gene (betaglobin) that was used as an internal control for PCR. We used the primers and protocols described by Quaresma et al. (2009). DNA was extracted using a Wizard® Genomic DNA Purification Kit (Promega, USA) and quantified by spectrophotometry. The amplification products were separated on 2.5% agarose gels (Invitrogen, USA), stained with ethidium bromide, visualized under UV light, and photographed using an LPIX apparatus (Loccus Biotecnologia, São Paulo, Brazil).
2. Materials and methods 3. Results
2.1. Dogs
Using culture-based analysis, parasites were detected in 23 dogs, including T. caninum in fragments of healthy skin from 11 dogs and L. chagasi in fragments of healthy skin from 8 dogs and in cutaneous lesions from 4 dogs. Using the 18S rDNA PCR assay, positive results were obtained for 35 dogs. Among these, 25 samples showed an amplification pattern similar to that of T. caninum (including all 11 that showed a positive culture test) and 10 samples showed an amplification pattern similar to that of L. chagasi. Sequencing of the PCR products confirmed the results obtained by electrophoretic analysis of the 18S rDNA amplification patterns. The samples identified as T. caninum showed 100% similarities with T. caninum sequences retrieved from GenBank (accession number: GU385824–GU385826, JF951431 and JF907507–JF907538). Likewise, the samples identified as L. chagasi showed 100% similarities with L. chagasi sequence. PCR amplification of kDNA yielded positive results for Leishmania infection for 14 dogs. Of these, 2 dogs showed negative culture results while 12 had positive results for L. chagasi, including 4 with negative 18S rDNA PCR results. Amplification products (118 bp) were obtained for the internal PCR control (beta-globin) for all the samples tested.
Two hundred and twenty-nine dogs were included in the study; the animals were selected during a serological survey for leishmaniasis, which was performed in the municipality of Rio de Janeiro (Figueiredo et al., 2010). The dogs were mechanically restrained and subjected to sedation with ketamine (10 mg/kg) associated with acepromazine (0.2 mg/kg). Following a clinical examination, two fragments of about 3 mm of healthy skin were collected for culture and PCR assays. This study was approved by the Ethics Committee on Animal Use of the Oswaldo Cruz Foundation (license L-017/06). 2.2. Culture and molecular assays Culture-based assays were performed according to methods described elsewhere (Madeira et al., 2009). For the samples from which parasites were isolated, we employed multilocus enzyme electrophoresis (MLEE; promastigote forms) or PCR/sequencing (epimastigote forms) for etiological characterization as described by Almeida et al. (2011). Three different PCR assays were carried out on tissue samples: The first targeted a partial sequence of the
Table 1 Results obtained from culture and molecular tools (PCR and sequencing) using skin fragments collected from 229 dogs in Rio de Janeiro, Brazil. In vitro isolation
PCR 18S rDNA
Sequencing 18S rDNA
Trypanosoma caninum (n = 11) Leishmania chagasi (n = 12) Negative (n = 206)
11 8 16
11 0 14
0 8 2
0 12 2
Total
35
25
10
14
Trypanosoma caninum
PCR kDNA Leishmania chagasi
A.G.S. Pinto et al. / Veterinary Parasitology 205 (2014) 343–346
The results obtained using the different approaches are summarized in Table 1. 4. Discussion Although many aspects of T. caninum are not well understood, certain characteristics have been established, as its exclusive isolation from healthy skin fragments, an unusual feature of the Trypanosoma genus. In our study, PCR-based assays were found to be more sensitive than culture tests for the identification of T. caninum, yielding 25 positive results compared to 11 cases identified by culture-based analysis. Similar results were obtained for L. chagasi infection, as the specific PCR and culture assays identified 14 and 12 infected animals, respectively. Thus, the PCR assay performed in this study is a potentially important resource for the diagnosis of T. caninum infections. Furthermore, our results suggest that the prevalence of T. caninum infection may be greater than that previously reported in studies using culture assays as a diagnostic tool (Almeida et al., 2011). The choice of skin specimens for our analysis was based on previous reports indicating that the skin is the only site in which T. caninum has been detected (Madeira et al., 2014). Notably, all of the skin samples that were found to be positive for T. caninum by culture-based isolation were also positive in the 18S rDNA PCR assay; this finding supports the efficacy of the PCR assay. Another important observation was that PCR inhibition was not seen for any of the samples analyzed. The amplification pattern and sequencing of the 18S rDNA PCR products showed that 10 dogs were positive for L. chagasi. For 8 of these, positive results were also obtained for L. chagasi via the culture assay, while negative culture results were obtained for 2. However, all 12 dogs that showed positive results for L. chagasi in the culture assay also showed positive results when the skin samples were analyzed using a Leishmania-specific kDNA target. The large copy number of minicircles present in the kinetoplast may explain these results (Lachaud et al., 2002). Furthermore, kDNA is a more specific target than 18S rDNA. It is also important to note that, for the 4 animals whose 18S rDNA PCR results were negative, the detection of L. chagasi was obtained by culture of using cutaneous lesion fragments collected from these animals. Improved tools for the diagnosis of T. caninum are necessary, particularly for differentiation between T. caninum and L. chagasi. In this study, differences in the 18S rDNA PCR amplification patterns of these 2 species were evident on performing gel electrophoresis; however, sequencing was necessary to confirm the results. Although the sequencing step of this approach is a limiting factor for mass screening, it could be a beneficial alternative to culture-based diagnosis, particularly in regions where both agents co-exist and when serological testing is not available. Many studies have shown cross-reactivity in tests used for the diagnosis of CVL; however, when we analyzed the serological results of the animals studied herein, we observed that the T. caninum infected dogs do not cross react with CVL tests in Rio
345
de Janeiro state (data not shown). This result suggests that serology should continue to be the mainstay for leishmaniasis infection screening in dogs. This study highlights the usefulness of PCR tools for determining the prevalence of T. caninum in Brazil, which has been reported in only a few studies to date. Our findings indicate that diagnostic and screening efforts should focus on molecular studies, particularly in regions where T. caninum has not yet been described. Thus far, culturebased testing has been the only tool used successfully for T. caninum diagnosis; however, our results demonstrate that molecular assays are more effective for this purpose and may be a useful diagnostic tool, particularly in regions endemic for both T. caninum and L. chagasi. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This study is part of the doctoral thesis of Andressa Guimarães de Souza Pinto at the Clinical Research of Infectious Diseases Program of the Evandro Chagas Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. The present study was partially financed by the National Council of Research Development (CNPq – Program PAPES VI, process 407700/2012-9) and Research Support Foundation of Rio de Janeiro State (FAPERJ – Program Young Scientist from our state, process E26/103.223/2011). MF Madeira and FB Figueiredo hold a grant for productivity in research. References Almeida, A.B.P.F., Sousa, V.R.F., Boa Sorte, E.C., Figueiredo, F.B., Paula, D.A.J., Pimentel, M.F., Dutra, V., Madeira, M.F., 2011. Use of parasitological culture to detect Leishmania (Leishmania) chagasi in naturally infected dogs. Vector Borne Zoonotic Dis. 11, 1555–1560. Alves, A.S., Mouta-Confort, E., Figueiredo, F.B., Oliveira, R.V.C., Schubach, A.O., Madeira, M.F., 2012. Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum. Res. Vet. Sci. 93, 1329–1333. Barros, J.H.S., Almeida, A.B.P.F., Figueiredo, F.B., Sousa, V.R.F., Fagundes, A., Pinto, A.G.S., Baptista, C., Madeira, M.F., 2012. Occurrence of Trypanosoma caninum in areas overlapping with leishmaniasis in Brazil: what is the real impact of canine leishmaniasis control? Trans. R. Soc. Trop. Med. Hyg. 106, 419–423. Degrave, W., Fernandes, O., Campbell, D., Bozza, M., Lopes, U., 1994. Use of molecular probes and detection and typing of Leishmania – a minirewiew. Mem. Inst. Oswaldo Cruz 89, 463–469. Figueiredo, F.B., Madeira, M.F., Nascimento, L.D., Abrantes, T.R., MoutaConfort, E., Passos, S.R.L., Schubach, T.M.P., 2010. Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples. Rev. Inst. Med. Trop. São Paulo 52, 193–196. Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J.P., Bastien, P., 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40, 210–215. Madeira, M.F., Sousa, M.A., Barros, J.H., Figueiredo, F.B., Fagundes, A., Schubach, A., de Paula, C.C., Faissal, B.N.S., Fonseca, T.S., Thoma, H.K., Marzochi, M.C.A., 2009. Trypanosoma caninum n. sp. (Protozooa: Kinetoplastida) isolated from intact skin of a domestic dog (Canis familiaris) captured in Rio de Janeiro, Brazil. Parasitology 136, 411–423. Madeira, M.F., Almeida, A.B.P.F., Barros, J.H., Oliveira, T.S.F., Sousa, V.R.F., Alves, A.S., Miranda, L.F.C., Schubach, A.O., Marzochi, M.C.A., 2014. Trypanosoma caninum, a new parasite described in dogs in Brazil: aspects of natural infection. J. Parasitol. 100, 231–234.
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Pinto, A.G.S., Schubach, T.M.P., Figueiredo, F.B., Fagundes, A., Barros, J.H.S., de Paula, C.C., Toma, H.K., Madeira, M.F., 2010. Isolation of Trypanosoma caninum in domestic dogs in Rio de Janeiro, Brazil. Parasitology 137, 1653–1660. Quaresma, P.F., Murta, S.M.F., Ferreira, E.C., Rocha-Lima, A.C.V.M., Xavier, A.A.P., Gontijo, C.M.F., 2009. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR. Acta Trop. 111, 289–294.
Silva, D.A., Madeira, M.F., Teixeira, A.C., Souza, C.M., Figueiredo, F.B., 2011. Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Vet. Parasitol. 179, 257–261. Smith, A., Clark, P., Averis, S., Lymbery, A.J., Wayne, A.F., Morris, K.D., Thompson, R.C., 2008. Trypanosomes in a declining species of threatened Australian marsupial, the brush-tailed bettong Bettongia penicillata (Marsupialia: Potoroidae). Parasitology 135, 1329–1335.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Evaluation of 18S rDNA PCR assay using skin fragments as a diagnostic test for Trypanosoma caninum A.G.S. Pinto a,b , H.K. Toma c , F.B. Figueiredo d , M.F. Madeira b,∗ a Programa de pós-graduac¸ão em Pesquisa Clínica em Doenc¸as Infecciosas, Instituto de Pesquisa Clínica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil b Laboratório de Vigilância em Leishmanioses, Instituto de Pesquisa Clínica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil c Laboratório de Diagnóstico Molecular e Hematologia, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Rio de Janeiro, RJ, Brazil d Laboratório de Pesquisa Clínica em Dermatozoonoses de Animais Domésticos, Instituto de Pesquisa Clínica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 7 February 2014 Received in revised form 23 July 2014 Accepted 26 July 2014 Keywords: Trypanosoma caninum PCR Dog Leishmaniasis
a b s t r a c t Trypanosoma caninum is a new species that has been recently identified in Brazil and infects domestic dogs. To date, no accurate diagnostic assays for this parasite have been established; thus, our aim was to evaluate more than one type of PCR for the diagnosis and molecular screening of T. caninum in 229 dogs living in Rio de Janeiro state. The tests were based on the amplification and sequencing of the 18S ribosomal DNA (rDNA) gene using healthy skin fragments. Additionally, PCR amplification of the kDNA minicircles region specific to the Leishmania genus was performed. The PCR results were compared with those of culture-based analysis performed with the same specimen. Using cultures, T. caninum and Leishmania chagasi were isolated from 11 and 12 dogs, respectively, whereas the 18S rDNA PCR assay detected parasitic infection in 35 dogs. Among these, 25 dogs showed an amplification pattern similar to T. caninum and 10 showed a pattern similar to L. chagasi; these results were confirmed by sequencing analysis. The kDNA PCR analysis showed that 14 dogs were positive for Leishmania infection. Of these, 2 dogs showed negative culture results and 12 were positive for L. chagasi, including 4 with negative 18S rDNA PCR results. Thus far, culture-based testing has been the only tool used successfully for T. caninum diagnosis. Our results demonstrate that 18S rDNA PCR-based test should be a useful diagnostic tool, particularly for distinguishing between T. caninum and L. chagasi infections in areas where these 2 parasites co-exist. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma caninum was initially identified in a dog co-infected with Leishmania braziliensis in the municipality of Rio de Janeiro, and was subsequently detected in other
∗ Corresponding author. Tel.: +55 21 3865 9541; fax: +55 21 38659541. E-mail address: fatima.madeira@ipec.fiocruz.br (M.F. Madeira). http://dx.doi.org/10.1016/j.vetpar.2014.07.028 0304-4017/© 2014 Elsevier B.V. All rights reserved.
regions of Brazil (Madeira et al., 2009; Barros et al., 2012). Despite attempts to isolate this parasite from blood and tissue samples, all 53 isolates of T. caninum described to date have been obtained from cultures of healthy skin samples from dogs (Barros et al., 2012). This strongly suggests that skin specimens could be used for other assays, such as PCR-based tests. Although certain epidemiological aspects are still unknown, the presence of T. caninum in regions where
344
A.G.S. Pinto et al. / Veterinary Parasitology 205 (2014) 343–346
canine visceral leishmaniasis (CVL) is endemic has been regarded as a possible confounding factor in the control of leishmaniasis (Alves et al., 2012). Previous studies have reported that dogs that were infected with T. caninum and were also seropositive for Leishmania were euthanized, in agreement with the CVL control measures established in Brazil; however, the presence of Leishmania chagasi (syn. Leishmania infantum) was not confirmed in those animals (Pinto et al., 2010; Silva et al., 2011). These findings highlight the importance of using accurate methods that can clearly distinguish Leishmania spp. infections from T. caninum infections, especially in areas where both parasites co-exist. T. caninum is found in various CVL endemic regions in Brazil, but data regarding its prevalence rates are limited, particularly in the municipality of Rio de Janeiro where most cases have been registered (Barros et al., 2012). However, culture-based testing has been the only tool used successfully for T. caninum diagnosis to date. Therefore, our study aimed to assess a PCR-based tool targeting 18S ribosomal DNA (rDNA) for the diagnosis and screening of T. caninum infection in domestic dogs living in the municipality of Rio de Janeiro, Brazil.
18S rDNA gene (18S rDNA PCR) and involved a nested assay according to a previously described protocol (Smith et al., 2008; Barros et al., 2012). The amplification products were purified using a QIAquick Purification Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The nucleotide sequences were processed using an automated sequencer (3730 DNA Analyzer; Applied Biosystems), edited by BioEdit software, and analyzed using the Basic Local Alignment Search Tool (BLAST). The second PCR assay, performed for all samples with positive culture and/or 18S rDNA PCR test results, targeted a conserved region of kDNA minicircles specific to the Leishmania genus, based on protocols described by Degrave et al. (1994). The third assay targeted a constitutive canine gene (betaglobin) that was used as an internal control for PCR. We used the primers and protocols described by Quaresma et al. (2009). DNA was extracted using a Wizard® Genomic DNA Purification Kit (Promega, USA) and quantified by spectrophotometry. The amplification products were separated on 2.5% agarose gels (Invitrogen, USA), stained with ethidium bromide, visualized under UV light, and photographed using an LPIX apparatus (Loccus Biotecnologia, São Paulo, Brazil).
2. Materials and methods 3. Results
2.1. Dogs
Using culture-based analysis, parasites were detected in 23 dogs, including T. caninum in fragments of healthy skin from 11 dogs and L. chagasi in fragments of healthy skin from 8 dogs and in cutaneous lesions from 4 dogs. Using the 18S rDNA PCR assay, positive results were obtained for 35 dogs. Among these, 25 samples showed an amplification pattern similar to that of T. caninum (including all 11 that showed a positive culture test) and 10 samples showed an amplification pattern similar to that of L. chagasi. Sequencing of the PCR products confirmed the results obtained by electrophoretic analysis of the 18S rDNA amplification patterns. The samples identified as T. caninum showed 100% similarities with T. caninum sequences retrieved from GenBank (accession number: GU385824–GU385826, JF951431 and JF907507–JF907538). Likewise, the samples identified as L. chagasi showed 100% similarities with L. chagasi sequence. PCR amplification of kDNA yielded positive results for Leishmania infection for 14 dogs. Of these, 2 dogs showed negative culture results while 12 had positive results for L. chagasi, including 4 with negative 18S rDNA PCR results. Amplification products (118 bp) were obtained for the internal PCR control (beta-globin) for all the samples tested.
Two hundred and twenty-nine dogs were included in the study; the animals were selected during a serological survey for leishmaniasis, which was performed in the municipality of Rio de Janeiro (Figueiredo et al., 2010). The dogs were mechanically restrained and subjected to sedation with ketamine (10 mg/kg) associated with acepromazine (0.2 mg/kg). Following a clinical examination, two fragments of about 3 mm of healthy skin were collected for culture and PCR assays. This study was approved by the Ethics Committee on Animal Use of the Oswaldo Cruz Foundation (license L-017/06). 2.2. Culture and molecular assays Culture-based assays were performed according to methods described elsewhere (Madeira et al., 2009). For the samples from which parasites were isolated, we employed multilocus enzyme electrophoresis (MLEE; promastigote forms) or PCR/sequencing (epimastigote forms) for etiological characterization as described by Almeida et al. (2011). Three different PCR assays were carried out on tissue samples: The first targeted a partial sequence of the
Table 1 Results obtained from culture and molecular tools (PCR and sequencing) using skin fragments collected from 229 dogs in Rio de Janeiro, Brazil. In vitro isolation
PCR 18S rDNA
Sequencing 18S rDNA
Trypanosoma caninum (n = 11) Leishmania chagasi (n = 12) Negative (n = 206)
11 8 16
11 0 14
0 8 2
0 12 2
Total
35
25
10
14
Trypanosoma caninum
PCR kDNA Leishmania chagasi
A.G.S. Pinto et al. / Veterinary Parasitology 205 (2014) 343–346
The results obtained using the different approaches are summarized in Table 1. 4. Discussion Although many aspects of T. caninum are not well understood, certain characteristics have been established, as its exclusive isolation from healthy skin fragments, an unusual feature of the Trypanosoma genus. In our study, PCR-based assays were found to be more sensitive than culture tests for the identification of T. caninum, yielding 25 positive results compared to 11 cases identified by culture-based analysis. Similar results were obtained for L. chagasi infection, as the specific PCR and culture assays identified 14 and 12 infected animals, respectively. Thus, the PCR assay performed in this study is a potentially important resource for the diagnosis of T. caninum infections. Furthermore, our results suggest that the prevalence of T. caninum infection may be greater than that previously reported in studies using culture assays as a diagnostic tool (Almeida et al., 2011). The choice of skin specimens for our analysis was based on previous reports indicating that the skin is the only site in which T. caninum has been detected (Madeira et al., 2014). Notably, all of the skin samples that were found to be positive for T. caninum by culture-based isolation were also positive in the 18S rDNA PCR assay; this finding supports the efficacy of the PCR assay. Another important observation was that PCR inhibition was not seen for any of the samples analyzed. The amplification pattern and sequencing of the 18S rDNA PCR products showed that 10 dogs were positive for L. chagasi. For 8 of these, positive results were also obtained for L. chagasi via the culture assay, while negative culture results were obtained for 2. However, all 12 dogs that showed positive results for L. chagasi in the culture assay also showed positive results when the skin samples were analyzed using a Leishmania-specific kDNA target. The large copy number of minicircles present in the kinetoplast may explain these results (Lachaud et al., 2002). Furthermore, kDNA is a more specific target than 18S rDNA. It is also important to note that, for the 4 animals whose 18S rDNA PCR results were negative, the detection of L. chagasi was obtained by culture of using cutaneous lesion fragments collected from these animals. Improved tools for the diagnosis of T. caninum are necessary, particularly for differentiation between T. caninum and L. chagasi. In this study, differences in the 18S rDNA PCR amplification patterns of these 2 species were evident on performing gel electrophoresis; however, sequencing was necessary to confirm the results. Although the sequencing step of this approach is a limiting factor for mass screening, it could be a beneficial alternative to culture-based diagnosis, particularly in regions where both agents co-exist and when serological testing is not available. Many studies have shown cross-reactivity in tests used for the diagnosis of CVL; however, when we analyzed the serological results of the animals studied herein, we observed that the T. caninum infected dogs do not cross react with CVL tests in Rio
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de Janeiro state (data not shown). This result suggests that serology should continue to be the mainstay for leishmaniasis infection screening in dogs. This study highlights the usefulness of PCR tools for determining the prevalence of T. caninum in Brazil, which has been reported in only a few studies to date. Our findings indicate that diagnostic and screening efforts should focus on molecular studies, particularly in regions where T. caninum has not yet been described. Thus far, culturebased testing has been the only tool used successfully for T. caninum diagnosis; however, our results demonstrate that molecular assays are more effective for this purpose and may be a useful diagnostic tool, particularly in regions endemic for both T. caninum and L. chagasi. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This study is part of the doctoral thesis of Andressa Guimarães de Souza Pinto at the Clinical Research of Infectious Diseases Program of the Evandro Chagas Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. The present study was partially financed by the National Council of Research Development (CNPq – Program PAPES VI, process 407700/2012-9) and Research Support Foundation of Rio de Janeiro State (FAPERJ – Program Young Scientist from our state, process E26/103.223/2011). MF Madeira and FB Figueiredo hold a grant for productivity in research. References Almeida, A.B.P.F., Sousa, V.R.F., Boa Sorte, E.C., Figueiredo, F.B., Paula, D.A.J., Pimentel, M.F., Dutra, V., Madeira, M.F., 2011. Use of parasitological culture to detect Leishmania (Leishmania) chagasi in naturally infected dogs. Vector Borne Zoonotic Dis. 11, 1555–1560. Alves, A.S., Mouta-Confort, E., Figueiredo, F.B., Oliveira, R.V.C., Schubach, A.O., Madeira, M.F., 2012. Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum. Res. Vet. Sci. 93, 1329–1333. Barros, J.H.S., Almeida, A.B.P.F., Figueiredo, F.B., Sousa, V.R.F., Fagundes, A., Pinto, A.G.S., Baptista, C., Madeira, M.F., 2012. Occurrence of Trypanosoma caninum in areas overlapping with leishmaniasis in Brazil: what is the real impact of canine leishmaniasis control? Trans. R. Soc. Trop. Med. Hyg. 106, 419–423. Degrave, W., Fernandes, O., Campbell, D., Bozza, M., Lopes, U., 1994. Use of molecular probes and detection and typing of Leishmania – a minirewiew. Mem. Inst. Oswaldo Cruz 89, 463–469. Figueiredo, F.B., Madeira, M.F., Nascimento, L.D., Abrantes, T.R., MoutaConfort, E., Passos, S.R.L., Schubach, T.M.P., 2010. Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples. Rev. Inst. Med. Trop. São Paulo 52, 193–196. Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J.P., Bastien, P., 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40, 210–215. Madeira, M.F., Sousa, M.A., Barros, J.H., Figueiredo, F.B., Fagundes, A., Schubach, A., de Paula, C.C., Faissal, B.N.S., Fonseca, T.S., Thoma, H.K., Marzochi, M.C.A., 2009. Trypanosoma caninum n. sp. (Protozooa: Kinetoplastida) isolated from intact skin of a domestic dog (Canis familiaris) captured in Rio de Janeiro, Brazil. Parasitology 136, 411–423. Madeira, M.F., Almeida, A.B.P.F., Barros, J.H., Oliveira, T.S.F., Sousa, V.R.F., Alves, A.S., Miranda, L.F.C., Schubach, A.O., Marzochi, M.C.A., 2014. Trypanosoma caninum, a new parasite described in dogs in Brazil: aspects of natural infection. J. Parasitol. 100, 231–234.
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