Applications of polymerase chain reaction for the detection of equine Leishmania sp. infection

Taiane Acunha Escobar, Gabriela Döwich, Letícia Carvalho Cantele, Geórgia Camargo Góss, Marcelo Lameiro Porciúncula, Carla Teixeira Leite, Luísa Zuravski, Claudia Acosta Duarte, Irina Lubeck

Abstract


Leishmaniasis is a neglected zoonotic disease caused by a variety of pathogenic Leishmania species. In the New World, especially in Brazil, visceral leishmaniasis (VL) is caused by Leishmania infantum. The pathogen can infect several animal species including dogs, foxes, rodents, primates, felines, equines and humans. Dogs act as the primary domestic reservoirs. This study aimed to use polymerase chain reaction (PCR) for detecting Leishmania infection in horses living in a canine visceral leishmaniasis (CVL) endemic region. DNA samples from horse peripheral blood were used to perform PCR. Templates were amplified using primers for the kinetoplast DNA (kDNA) minicircles, which were able to detect different species of Leishmania. In addition, primers for internal transcribed spacer (ITS) of ribosomal DNA were used for detection of Trypanosomatidae sp. Amongst the 75 (39%) positive PCR samples from total 192 samples, 21 samples were positive for kDNA and 63 samples were positive for either ITS, ITS1, or ITS2 gene markers. The kDNA PCR and sequencing allowed the detection of L. infantum in horse blood samples. To our knowledge, this is the first report of equine infection with L. infantum in Southern Brazil. These results proved that L. infantum could also infect horses in addition to humans and dogs, as well as in European countries. This conclusion emphasizes the urgent need to follow up investigation of the infection in these animals.

Keywords


Leishmania infantum; Diagnostic methods; Infection; Horses.

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References


Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet,… Kasbari, M. (2017). Leishmania infections: molecular targets and diagnosis. Molecular Aspects of Medicine, 57, 1-29. doi: 10.1016/j.mam.2016.11.012

Almeida, A. B., Sousa, V. R., Gasparetto, N. D., Silva, G. F. da, Figueiredo, F. B., Dutra, V., . . . Madeira, M. F. (2013). Canine visceral leishmaniasis: diagnostic approaches based on polymerase chain reaction employing different biological samples. Diagnostic Microbiology and Infectious Disease, 76(3), 321-324. doi: 10.1016/j.diagmicrobio.2013.03.017

Belo, V. S., Werneck, G. L., Barbosa, D. S., Simões, T. C., Nascimento, B. W. L., Silva, E. S. da, & Struchiner, C. J. (2013). Correction: factors associated with visceral leishmaniasis in the americas: a systematic review and meta-analysis. PLoS Neglected Tropical Diseases, 7(5). doi: 10.1371/annotation/83856044-8747-4d93-9a1e-64f16bb60c07

Benassi, J. C., Benvenga, G. U., Ferreira, H. L.; Pereira, V. F., Keid, L. B., Soares, R.,… Oliveira, T. M. F.S. (2017). Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR. Experimental Parasitology, 177, 93-97. doi: 10.1016/j.exppara.2017.04.004

Bigeli, J. G., Oliveira Júnior, W. P. de, & Teles, N. M. M. (2012). Diagnosis of Leishmania (Leishmania) chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas, state of Tocantins, Brazil. Revista da Sociedade Brasileira de Medicina Tropical, 45(1),18-23. doi: 10.1590/S0037-86822012000100005

Ministério da Saúde. (2014). Manual de vigilância e controle da leishmaniose visceral. Brasília: Ministério da Saúde - Divisão de Vigilância Epidemiológica.

Brito, C. F. P. C., & Pita-Pereira, D. (2014). Diagnóstico molecular de Leishmania spp. em Flebótomos provenientes de áreas de ocorrência de leishmanioses. In F. da Conceição-Silva & C. R. Alves. Leishmanioses do continente americano (Chap. 13, pp. 217-231). Rio de Janeiro: Editora FIOCRUZ.

Cortes, S.; Rolão, N.; Ramada, J., & Campino, L. (2004). PCR as a rapid and sensitive tool in the diagnosis of human and canine leishmaniasis using Leishmania donovani sl-specific kinetoplastid primers. Transactions of the Royal Society of Tropical Medicine and Hygiene, 98(1), 12-17. doi: 10.1016/S0035-9203(03)00002-6

Dantas-Torres, F. (2009). Canine leishmaniosis in South America. Parasites & Vectors, 2(1), S1. doi: 10.1186/1756-3305-2-S1-S1

Deboni, S. C., Barbosa, M., & Ramos, R. R. (2011). Leishmaniose Visceral no Rio Grande do Sul. Boletim Epidemiológico, 13 (1), 1-8. Retrived from: https://www.cevs.rs.gov.br/upload/arquivos/ 201812/27124724-n-1-marco.pdf

Echchakery, M., Chicharro, C., Boussaa, S., Nieto, J., Carrillo, E., Sheila, O.,… Boumezzough, A. (2017). Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco. Parasites & Vectors, 10(1), 454-462. doi: 10.1186/s13071-017-2398-8

El Tai, N., Osman, O., El Fari, M., Presber, W., & Schönian, G. (2000). Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Transactions of the Royal Society of Tropical Medicine and Hygiene, 94(5), 575-579. doi: 10.1016/S0035-9203(00)90093-2

El Tai, N. O., El Fari, M., Mauricio, I., Miles, M. A., Oskam, L., El Safi, S. H.,… Schönian, G. (2001). Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Experimental Parasitology, 97(1), 35-44. doi: 10.1006/expr.2001.4592

Escobar, T. A., Döwich, G., Zuravski, L., Cantele, L. C., Duarte, C. A., & Lübeck, I. (2018). Risk factors associated to canine visceral leishmaniasis in Uruguaiana city, Brazil. Semina: Ciências Agrárias, 39(1), 211-219. Retrived from http://dx.doi.org/10.5433/1679-0359.2018v39n1p211

Feitosa, F. L. F., Leal, J., Mendes, L. C. N., Peiro, J. R., Perri, S. H. V., Lima, V. M. F. de,... Marcondes, M. (2012). Estudo soroepidemiológico de leishmaniose em equinos na região de Araçatuba-SP, Brasil, área endêmica para leishmaniose visceral. Brazilian Journal of Veterinary Research and Animal Science, 49(6), 500-502. Retrived from http://dx.doi.org/10.11606/issn.1678-4456.v49i6p500-502

Fernández-Bellon, H., Solano-Gallego, L., Bardagí, M., Alberola, J., Ramis, A., & Ferrer, L. (2006). Immune response to Leishmania infantum in healthy horses in Spain. Veterinary Parasitology, 135(2),181-185. Retrived from https://dx.doi.org/1016/j.vetpar.2005.09.007

Figueiredo, F., & Madeira, M. de F. (2014). Os parasitos e a questão da infecção em animais domésticos e domiciliados. In F. da Conceição-Silva & C. R. Alves. Leishmanioses do continente americano (Chap. 15, pp. 259-273). Rio de Janeiro: Editora FIOCRUZ.

Gama, A., Elias, J., Ribeiro, A. J., Alegria, N., Schallig, H. D., Silva, F.,... Cotovio, M. (2014). Cutaneous leishmaniosis in a horse from northern Portugal. Veterinary Parasitology, 200(1), 189-192. Retrived from https://dx.doi.org/10.1016/j.vetpar.2013.12.005

Koehler, K., Stechele, M., Hetzel, U., Domingo, M., Schönian, G., Zahner, H., & Burkhardt, E. (2002). Cutaneous leishmaniosis in a horse in southern Germany caused by Leishmania infantum. Veterinary Parasitology, 109(1), 9-17. doi: 10.1016/S0304-4017(02)00246-7

Kuhls, K., Mauricio, I. L., Pratlong, F., Presber, W., & Schönian, G. (2005). Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex. Microbes and Infection, 7(11-12), 1224-1234. doi: doi.org/10.1016/j.micinf.2005.04.009

Lainson, R., & Rangel, E. F. (2005). Lutzomyia longipalpis and the eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil: a review. Memórias do Instituto Oswaldo Cruz, 100(8), 811-827. doi: 10.1590/S0074-02762005000800001

le Fichoux, Y., Quaranta, J.-F., Aufeuvre, J.-P., Lelievre, A., Marty, P., Suffia, I.,… Kubar, J. (1999). Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. Journal of Clinical Microbiology, 37(6), 1953-1957.

Lombardi, M. C., Turchetti, A. P., Tinoco, H. P., Pessanha, A. T., Soave, S. A., Malta, M. C.,… Santos, R. L. (2014). Diagnosis of Leishmania infantum infection by polymerase chain reaction in wild mammals. Pesquisa Veterinária Brasileira, 34(12), 1243-1246. doi: 10.1590/S0100-736X2014001200017

Lombardo, G., Pennisi, M. G., Lupo, T., Chicharro, C., & Solano-Gallego, L. (2014). Papular dermatitis due to Leishmania infantum infection in seventeen dogs: diagnostic features, extent of the infection and treatment outcome. Parasites & Vectors 7(1), 120-131. doi: 10.1186/1756-3305-7-120

Lopes, E., Sevá, A., Ferreira, F., Nunes, C., Keid, L., Hiramoto, R., . . . Galvis-Ovallos, F. (2017). Serological and molecular diagnostic tests for canine visceral leishmaniasis in Brazilian endemic area: one out of five seronegative dogs are infected. Epidemiology & Infection, 145(12), 2436-2444. Retrived from https://doi-org.ez96.periodicos.capes.gov.br/10.1017/S0950268817001443

Lukeš, J., Guilbride, D. L., Votýpka, J., Zíková, A., Benne, R., & Englund, P. T. (2002). Kinetoplast DNA network: evolution of an improbable structure. Eukaryotic cell, 1(4), 495-502. doi: 10.1128/EC.1.4.495-502.2002

Macedo-Silva, V. P., Martins, D. R., Queiroz, P. V. S. de., Pinheiro, M. P. G., Freire, C. C., Queiroz, J. W., . . . Jeronimo, S. M. (2014). Feeding preferences of Lutzomyia longipalpis (Diptera: Psychodidae), the sand fly vector, for Leishmania infantum (Kinetoplastida: Trypanosomatidae). Journal of Medical Entomology 51(1), 237-244. doi: 10.1603/ME12131

Müller, N., Hentrich, B., Frey, C. F., & Welle, M. (2015). Quantitative PCR for the diagnosis of cutaneous leishmaniasis from formalin-fixed and paraffin-embedded skin sections. Molecular and Cellular Probes, 29(6), 507-510. doi: 10.1016/j.mcp.2015.09.008

Müller, N., Welle, M., Lobsiger, L., Stoffel, M. H., Boghenbor, K. K., Hilbe, M.,… Von Bomhard, W. (2009). Occurrence of Leishmania sp. in cutaneous lesions of horses in Central Europe. Veterinary Parasitology, 166(3), 346-351. doi: 10.1016/j.vetpar.2009.09.001

Pandey, K., Pant, S., Kanbara, H., Shuaibu, M. N., Mallik, A. K., Pandey, B. D.,… Yanagi, T. (2008). Molecular detection of Leishmania parasites from whole bodies of sandflies collected in Nepal. Parasitology Research, 103(2), 293-297. doi: 10.1007/s00436-008-0967-7

Pita-Pereira, D., Lins, R., Oliveira, M. P., Lima, R. B., Pereira, B. A., Moreira, O. C.,... Britto, C. (2012). SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniasis. Parasites & Vectors, 5(1), 5-15. doi: 10.1186/1756-3305-5-15

Sambrook, J., & Russel, D. W. (2001). Molecular cloning: a laboratory manual (Rev. ed.). (Chap. 1, pp. 21-27). New York: Cold Spring Harbor: Cold spring harbor laboratory press.

Schönian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, H. D., Presber, W., & Jaffe, C. L. (2003). PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagnostic Microbiology and Infectious Disease, 47(1), 349-358.

Schönian, G., Schnur, L., El Fari, M., Oskam, L., Kolesnikov, A. A., Sokolowska-Köhler, W., & Presber, W. (2001). Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods. Transactions of the Royal Society of Tropical Medicine and Hygiene, 95(2), 217-224. doi: 10.1016/S0035-9203(01)90173-7

Silva, A. F. da, Damasceno, Á. R. A., Prado, W. S., Caldeira, R. D., Sampaio-Junior, F. D., Farias, D. M. de, . . . G., Scofield, A. (2017). Leishmania infantum infection in dogs from maroon communities in the Eastern Amazon. Ciência Rural, 47, e20160025. doi: 10.1590/0103-8478cr20160025

Silva, M. A. L. da, Medeiros, R. A., Brandão-Filho, S., Melo, F. L., & Medeiros, Z. (2010). Alvos moleculares utilizados em PCR para o diagnóstico da leishmaniose visceral humana. Revista Eletrônica de Farmácia, 7(3), 1-15. doi: 10.5216/ref.v7i3.12947

Silva, R. C., Richini-Pereira, V. B., Kikuti, M., Marson, P. M., & Langoni, H. (2017). Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers. Veterinary Quarterly, 37(1), 23-30. doi: 10.1080/01652176.2016.1252073

Soares, I. R., Silva, S. O., Moreira, F. M., Prado, L. G., Fantini, P., Maranhão, R. de P. A.,... Palhares, M. S. (2013). First evidence of autochthonous cases of Leishmania (Leishmania) infantum in horse (Equus caballus) in the Americas and mixed infection of Leishmania infantum and Leishmania (Viannia) braziliensis. Veterinary Parasitology, 197(3), 665-669. doi: 10.1016/j.vetpar.2013.06.014

Solano-Gallego, L., Fernàndez-Bellon, H., Serra, P., Gállego, M., Ramis, A., Fondevila, D., & Ferrer, L. (2003). Cutaneous leishmaniosis in three horses in Spain. Equine Veterinary Journal, 35(3), 320-323. R doi: 10.2746/042516403776148336:

Solcà, M. da S., Guedes, C. E. S., Nascimento, E. G., Oliveira, G. G. de S., Santos, W. L. C. dos, Fraga, D. B. M., & Veras, P. S. T. (2012). Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs. Veterinary Parasitology, 184(2-4), 133-140. doi: 10.1016/j.vetpar.2011.08.026

Souza, N. P., Almeida, A. do B. P. F. de, Freitas, T. P. T. de, Paz, R. C. R. da, Dutra, V., Nakazato, L., & Sousa, V. R. F. (2010). Leishmania (Leishmania) infantum chagasi em canídeos silvestres mantidos em cativeiro, no Estado de Mato Grosso. Revista da Sociedade Brasileira de Medicina Tropical, 43(3), 333-335. doi: 10.1590/S0037-86822010000300024

Truppel, J. H., Otomura, F., Teodoro, U., Massafera, R., Costa-Ribeiro, M. C. V. da, Catarino, C. M.,... Thomaz-Soccol, V. (2014). Can equids be a reservoir of Leishmania braziliensis in endemic areas? PLoS ONE, 9(4), e93731. doi: 10.1371/journal.pone.0093731




DOI: http://dx.doi.org/10.5433/1679-0359.2020v41n1p199

Semina: Ciênc. Agrár.
Londrina - PR
E-ISSN 1679-0359
DOI: 10.5433/1679-0359
E-mail: semina.agrarias@uel.br
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