SciELO - Scientific Electronic Library Online

vol.22 issue4Use of a negative binomial distribution to describe the presence of Sphyrion laevigatum in Genypterus blacodesA case of nasal myiasis due to Oestrus ovis (Diptera: Oestridae) in a llama (Lama glama) author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand




Related links


Revista Brasileira de Parasitologia Veterinária

On-line version ISSN 1984-2961

Rev. Bras. Parasitol. Vet. vol.22 no.4 Jaboticabal Oct./Dec. 2013 

Research Note

Molecular and parasitological detection of Leishmania spp. in a dipteran of the species Tabanus importunus

Detecção molecular e parasitológica de Leishmania spp. em díptero da espécie Tabanus importunus

Willian Marinho Dourado Coelho1  * 

Katia Denise Saraiva Bresciani1 

1Departamento de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária de Araçatuba - FMVA, Universidade Estadual Paulista - UNESP, Araçatuba, SP, Brasil


Leishmaniasis is an important chronic zoonosis caused by protozoa of the genus Leishmania spp. The major vectors of this protozoosis are sand flies, and Lutzomyia longipalpis is considered the main species implicated in the transmission of American Visceral Leishmaniasis in Brazil. The presence of the parasite's deoxyribonucleic acid (DNA) in ectoparasites such as ticks and fleas has prompted speculations about the existence of new vectors in the cycle of leishmaniasis. The aim of this paper is to report the molecular detection of Leishmania spp. in a horse fly of the species Tabanus importunus which parasitized an oligosymptomatic dog infected with Leishmania spp. Molecular amplification of the protozoan's DNA in the head, thoracic region and abdomen of the tabanid tested positive for Leishmania complex. This is the first report of the presence of DNA from Leishmania spp. in dipterous insects of the species T. importunus.

Key words: Dogs; canine visceral leishmaniasis; horse fly; PCR; protozoan; vectors


A leishmaniose é uma importante zoonose, de caráter crônico, causada por protozoários do gênero Leishmania spp. Esta protozoose tem como principal vetor os flebotomíneos, sendo que, no Brasil, o Lutzomyia longipalpis é a principal espécie incriminada na transmissão da leishmaniose Visceral Americana. A presença do ácido desoxirribonucleico (DNA) do parasito em ectoparasitos, como carrapatos e pulgas, tem gerado especulações quanto a existência de novos vetores no ciclo da leishmaniose. Foi objetivo deste estudo relatar a detecção molecular de Leishmania spp. em uma mutuca da espécie Tabanus importunus que parasitava um cão oligossintomático infectado por Leishmania spp. A análise molecular amplificou o DNA do protozoário na cabeça, na região torácica e no abdomen do tabanídeo, resultando como positivo para complexo Leishmania. Este é o primeiro relato da presença de DNA de Leishmania spp. em insetos dipteros da espécie T. importunus.

Palavras-Chave: Cães; leishmaniose visceral canina; Mutuca; PCR; protozoário; vetores


Leishmaniasis are an important disease, with a range of clinical and epidemiological features, more frequently reported in regions where several species of sand flies exist, particularly the species of Lutzomyia longipalpis (LAURENTI et al., 2009; SOARES et al., 2010; COSTA et al., 2013).

Although leishmaniasis is transmitted by sand fly bites infection via other routes have been reported, including congenital transmission in dogs (ROSYPAL et al., 2005; DA SILVA et al., 2009) and blood transfusions (DE FREITAS et al., 2006; GOODNOUGH, 2013). Among other possibilities of transmission, researchers have reported the presence of DNA from this parasite in ixodids of the species Rhipicephalus sanguineus and in fleas of the species Ctenocephalides felis felis, suggesting that these arthropods may act positively in the epidemiology of this disease (OTRANTO; DANTAS-TORRES, 2010). Horse flies are hematophagous insects known to be important vectors of several etiologic agents that cause diseases, particularly trypanosomiasis (SILVA et al., 1996).

The aim of this paper is to report the molecular detection of Leishmania spp. in a horse fly of the species Tabanus importunus captured in the region of Andradina, state of São Paulo, Brazil.


An adult male dog of undefined breed, short-haired and predominantly black, was sent to the Veterinary Hospital of Andradina Educational College in the state of São Paulo (20.8961°, 51.37944°, 405 m altitude). This animal lived in a peri-urban zone in a house with a large yard shaded by fruit and ornamental trees, in the company of other animals such as exotic caged birds and chickens in a chicken run.

The dog was clinically characterized as oligosymptomatic for leishmaniasis (onychogryphosis, weight loss, lymphadenomegaly, muscle atrophy and dermatitis). During anamnesis at the animal's place of origin, a horse fly was observed sucking blood on the dog's left dorsal thoracic region.

In this municipality and surroundings, these tabanids frequently attack farm animals such as cattle, horses and goats, and parasitize dogs, nestlings in their nests, and even people.

The insect was captured by hand, stored in a plastic tube and frozen at −20 °C. The animal was diagnosed parasitologically as a carrier of Leishmania spp. infection, since numerous amastigote forms of Leishmania spp. were visible in the imprint of the right popliteal lymph node. The dog was euthanized according to Brazilian legislation, as per Resolution no. 1000/2012 of the Federal Council of Veterinary Medicine, and subjected to necropsy.

The dipteran collected from the animal was divided into three parts with a bistoury, and these parts were separated into aliquots and identified as follows: head (H1), thorax (T1) and abdomen (A1). When stored in the tube, the insect ejected a droplet of blood-like material from the posterior end of its abdomen (Figure 1). The tube was washed with 0.5 mL of 0.9% sterile sodium chloride solution and the sample was classified as washed (W1).

Figure 1.  Tabanus importunus infected by Leishmania spp. 

The insect parts were macerated in a porcelain crucible containing 0.5 mL of the same physiological solution. Similarly, the blood sample and the lymph node fragment from the dog were collected, divided into aliquots and stored at −20 °C for analysis by polymerase chain reaction (PCR).

The aliquots were subjected to molecular analysis by PCR, using oligonucleotides that amplify the conserved region of the kinetoplast (kDNA) minicircle, using the primers 13A (5′-GTG GGG GAG GGG CGT TCT -3′) e 13B (5′-ATT TTA CAC CAA CCC CCA GTT-3′) (RODGERS et al., 1990). These analyses were performed by an outsourced laboratory known for its accurate diagnosis of animal and human leishmaniasis.

Part of W1 was used to prepare the smear on a microscope slide. This material was fixed and stained using a "Panótico Rápido®" kit and was examined under 1000x magnification in a light microscope equipped with a flat achromatic lens; 300 microscope fields were examined.

The molecular analysis indicated that all the samples from the arthropod (H1, T1, A1 and W1) and from the dog were positive for Leishmania complex, and the reference value adopted was the negative result. Microscopic analysis of the smear from W1 showed amastigotes forms of Leishmania spp. (Figure 1).

Leishmaniasis is an important zoonosis whose cycle is classically characterized by the presence of dipterous vectors of the genus Lutzomyia (GALATI et al., 2003; KAMHAWI, 2006; SILVA et al., 2008). However, entomological studies carried out in different Brazilian municipalities did not confirm the presence of this phlebotomine in areas endemic to L. chagasi (DANTAS-TORRES, 2006). Thus, the possible existence of a new vector has been suggested in the epidemiology of this parasitosis (PAZ, 2010a).

Several researchers have mentioned the possibility that R. sanguineus acts as a vector of leishmaniasis (PAZ et al., 2010b), either through its bite (DANTAS-TORRES et al., 2010) or through the ingestion of infected ticks (COUTINHO et al., 2005). Similarly, fleas have been reported as vectors of Leishmania spp. (COUTINHO; LINARDI, 2007; PAZ, 2010a). However, the detection of Leishmania spp. DNA does not suffice to imply a species as vector (SAVANI et al., 2009).

In this sense, coinfection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi has already been found in a dog from the state of Mato Grosso do Sul (SAVANI et al., 2005).

In this region, the non-selective behavior of these tabanids for host species is worthy of note, as it is an endemic area for human and canine visceral leishmaniasis. This fact is worth highlighting because tabanids are considered important vectors of trypanosomiasis in animals (NUNES, 1996; FRANKE et al., 1994; HERRERA et al., 2004).

The epidemiology of leishmaniasis is a constantly expanding theme and the action of new vectors in the cycle of this disease must be considered. Thus, this paper offers the first report of the presence of amastigotes forms and DNA of Leishmania spp. in a horse fly of the species T. importunus parasitizing a dog that was a carrier of canine visceral leishmaniasis, suggesting the possibility that the dipteran of species Tabanus importunus act as mechanical vectors in the cycle of this zoonosis.


Costa LP, Dantas-Torres F, Da Silva FJ, Guimarães VCFV, Gaudêncio K, Brandão-Filho SP. Ecology of Lutzomyia longipalpis in an area of visceral leishmaniasis transmission in north-eastern Brazil. Acta Trop 2013; 126(2): 99-102. PMid:23369878. ]

Coutinho MTZ, Linardi PM. Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? Vet Parasitol 2007; 147(3-4): 320-325. PMid:17521814. ]

Coutinho MTZ, Bueno LL, Sterzik A, Fujiwara RT, Botelho JR, De Maria M, et al. Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol 2005; 128(1-2): 149-155. PMid:15725545. ]

Dantas-Torres F, Lorusso V, Testini G, DE Paiva-Cavalcanti M, Figueredo LA, Stanneck D, et al. Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy. Parasitol Res 2010; 106(4): 857-860. PMid:20127362. ]

Dantas-Torres F. Do any insects other than phlebotomine sand flies (Diptera: Psychodidae) transmit Leishmania infantum (Kinetoplastida: Trypanosomatidae) from dog to dog? Vet Parasitol 2006; 136(3-4): 379-380. PMid:16417969. ]

Da Silva SM, Ribeiro VM, Ribeiro RR, Tafuri WL, Melo MN, Michalick MSM. First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil. Vet Parasitol 2009; 166(1-2): 159-162. PMid:19733439. ]

De Freitas E, Melo MN, Da Costa-Val AP, Michalick MS. Transmission of Leishmania infantum via blood transfusion in dogs: potential for infection and importance of clinical factors. Vet Parasitol 2006; 137(1-2): 159-167. PMid:16414196. ]

Franke CR, Greiner M, Mehlitz D. Investigations on naturally occurring Trypanosoma evansi infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Poconé (Mato Grosso, Brazil). Acta Trop 1994; 58(2): 159-169. ]

Galati EAB, Nunes VLB, Cristaldo G, Da Rocha HC. Aspectos do comportamento da fauna flebotomínea (Diptera: Psychodidae) em foco de leishmaniose visceral e tegumentar na Serra da Bodoquena e area adjacente, Estado de Mato Grosso do Sul, Brasil. Rev Patol Trop 2003; 32(2): 235-261. [ Links ]

Goodnough LT. Blood management: transfusion medicine comes of age. Lancet 2013; 381(9880): 1791-1792. ]

Herrera HM, Dávila AMR, Norek A, Abreu UG, Souza SS, D'Andrea PS, et al. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet Parasitol 2004; 125(3-4): 263-275. PMid:15482883. ]

Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol 2006; 22(9): 439-445 PMid:16843727. ]

Laurenti MD, Silveira VM, Secundino MF, Corbett CE, Pimenta PP. Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild-caught Lutzomyia longipalpis. Parasitol Int 2009; 58(3): 220-226. PMid:19454323. ]

Nunes VLB. Protozoários tripanosomatídeos encontrados em animais domésticos e silvestres em área do Planalto e do Pantanal Sul-mato-grossense. Mem Inst Oswaldo Cruz 1996; 91(S): 27. [ Links ]

Otranto D, Dantas-Torres F. Fleas and ticks as vectors of Leishmania spp. to dogs: caution is needed. Vet Parasitol 2010; 168(1-2): 173-174. PMid:20022429. ]

Paz GF, Ribeiro MFB, De Magalhães DF, Sathler KPB, Morais MHF, Fiúza VOP, et al. Association between the prevalence of infestation by Rhipicephalus sanguineus and Ctenocephalides felis felis and the presence of anti-Leishmania antibodies: a case-control study in dogs from a Brazilian endemic area. Prev Vet Med 2010a; 97(2); 131-133. PMid:20869131. ]

Paz GF, Ribeiro MFB, Michlasky EM, Da Rocha Lima ACVM, França-Silva JC, et al. Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis. Parasitol Res 2010b; 106(2): 523-528. PMid:20012095. ]

Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as tool in the detection and diagnosis of Leishmania. Exp Parasitol 1990; 71(3): 267-275. ]

Rosypal AC, Troy GC, Zajac AM, Frank G, Lindsay DS. Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle. J Parasitol 2005; 91(4): 970-972. PMid:17089780. ]

Savani ESMM, Nunes VLB, Galati EAB, Castilho TM, Zampieri RA, Floeter-Winter LM. The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil. Vet Parasitol 2009; 160(1-2): 18-24. PMid:19062193. ]

Savani ESMM, Nunes VLB, Galati EAB, Castilho TM, De Araujo FS, Ilha IMN, et al. Occurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz 2005; 100(7): 739-741. ]

Silva EA, Andreotti R, Dias ES, Barros JC, Brazuna JC. Detection of Leishmania DNA in phlebotomines captured in Campo Grande, Mato Grosso do Sul, Brazil. Exp Parasitol 2008; 119(3): 343-348. PMid:18456262. ]

Silva RAMS, Silva JA, Schneider RC, Freitas J, Mesquita D, Mesquita T, et al. Outbreak of trypanosomiasis due to Trypanosoma vivax (Ziemann, 1905) in bovines of the Pantanal, Brazil. Mem Inst Oswaldo Cruz 1996; 91 (5): 561-562. ]

Soares MRA, Carvalho CC, Silva LA, Lima MSCS, Barral AMP, Rebêlo JMM, et al. Molecular analysis of natural infection of Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in Brazil. Cad Saúde Pública. 2010; 26(12): 2409-2413. PMid:21243235. ]

Received: April 29, 2013; Accepted: June 4, 2013

*Corresponding author: Willian Marinho Dourado, Coelho Departamento de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária de Araçatuba - FMVA, Universidade Estadual Paulista - UNESP, Rua Clóvis Pestana, 793, CEP 16050-680, Araçatuba, SP, Brasil, e-mail:

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License.