The transmission of Leishmania infantum chagasi by the bite of the Lutzomyia longipalpis to two different vertebrates

Parasites & Vectors The transmission of Leishmania infantum chagasi by the bite of the Lutzomyia longipalpis to two different vertebrates Nagila FC Secundino 0 Vanessa C de Freitas 0 Carolina C Monteiro 0 Ana-Clara AM Pires 0 Bruna A David 0 Paulo FP Pimenta 0 0 Laboratory of Medical Entomology of the Centro de Pesquisas Rene Rachou, Fundacao Oswaldo Cruz - MG. Av. Augusto Lima , 1715 Barro Preto, Belo Horizonte, 30190-002, Minas Gerais , Brazil Background: Sandflies are vectors of Leishmania, the causative agent of leishmaniasis in mammalian hosts, including humans. The protozoan parasite is transmitted by the sandfly bite during salivation that occurs at the moment of blood feeding. The components of vector saliva include anticlotting and vasodilatory factors that facilitate blood flow and immunomodulatory factors that inhibit wound healing and quell the immune response. Not surprisingly, these factors also play important roles in the establishment of Leishmania infection. To date, the majority of knowledge that has been generated regarding the process of Leishmania infection, including L. infantum chagasi transmission has been gathered by using intradermal or subcutaneous inoculation of purified parasites. Findings: This study presents the establishment of a transmission model of Leishmania infantum chagasi by the bite of Lutzomyia longipalpis, the vector of American visceral leishmaniasis. The parasites were successfully transmitted by infected sandfly bites to mice and hamsters, indicating that both animals are good experimental models. The L. infantum chagasi dose that was transmitted in each single bite ranged from 10 to 10, 000 parasites, but 75% of the sandflies transmitted less than 300 parasites. Conclusions: The strategy for initiating infection by sandfly bite of experimental animals facilitates future investigations into the complex and dynamic mechanisms of visceral leishmaniasis. It is important to elucidate the transmission mechanism of vector bites. This model represents a useful tool to study L. infantum chagasi infection transmitted by the vector. transmission; bite; leishmaniasis; Lutzomyia longipalpis - Findings Despite the fact that Leishmania is transmitted exclusively by sandfly vectors, a reproducible animal model of Leishmania infection transmitted by sandfly bite was only described in the year 2000 [1]. Early investigations showed that infected P. papatasi sandflies can release from 0 to over 1,000 L. major promastigotes through their proboscides by forced feeding [2]. However, a transmission model by bite with P. duboscqui infected with L. major found that the parasite numbers inoculated in the host skin by one insect vector alone could vary from 10 to 100,000 [3]. Considering the New World species of Leishmania, using a feeding device of chick skin membrane over culture medium showed that L. longipalpis sandflies infected with Leishmania mexicana (an unnatural vector/parasite pair) expelled an average of 1,000 parasites per fly [4]. Thus, even though L. longipalpis is the vector of L. infantum chagasi in nature, it is permissive in the laboratory to infection with other Leishmania species. In consideration of the continuing prevalence of American visceral leishmaniasis and the paucity of related studies in the literature, our goal was to develop an L. infantum chagasi transmission model by bite, using its proven vector, L. longipalpis. In addition, we aimed to define the amount of parasites that are directly expelled into the mammalian host, which has never before been carried out. To evaluate the sandfly infection, colonized two- to four-day-old L. longipalpis females (Lapinha Cave strain) were infected by feeding through a chick skin membrane (feeding device) on mouse blood containing 4106/mL L. infantum chagasi (MHOM/BR/70/BH46) promastigotes. After two, six, nine and fourteen days, the midguts of infected flies were dissected and the parasite load was estimated by hemocytometer counting. For Transmission by bite, fourteen-day infected flies were transferred to small plastic vials (3-dram volume, 4.8-cm height, 1.8-cm diameter) covered at one end with a 0.25-mm nylon mesh. Balb/C mice and hamsters were anesthetized by Thiopental injection. Clamps were used to hold the mesh end of each vial flat against the animals ears so that the fly had access to the ear skin for feeding over a period of 1-2 h in the dark. The animals were then euthanized and the exposed ears were dissected for testing the parasite presence. One infected sandfly was used for each of the transmission experiments (Figure 1). All animals were maintained at the Animal Care Facility of the FIOCRUZ-MG under specific pathogen-free conditions and were used in accordance to a study protocol approved by the FIOCRUZ Ethical Committee for Animal Use (CEUA; license number LW30/10). Exposed-bite animal ears were processed for DNA extraction and real-time PCR as described elsewhere [3]. Parasite quantification was performed by automatic comparison with the specific set of standard samples prepared in parallel to each set of test samples. The number of L. infantum chagasi in each sample was summarized as the mean of the two median values from the three reactions in each run. Twenty-eight or 30 ears from each group (mouse or hamster) were processed. A total of 640 L. longipalpis females from 800 individuals ingested the infected bloodmeal containing L. infantum chagasi promastigotes. A group of 300 sandflies from this collection were dissected and evaluated up to 14 days after infection. Seventy-two hours after the infective bloodmeal, 100% of the sandflies were positive for viable promastigotes, with an average of 14,000 promastigotes per midgut. At six days post-infection, there was an approximate 70% reduction in parasite growth and survival (p = 0.0009), with an infection average of 3, 000 parasites per sandfly; this change may reflect loss of parasites due to fly defecation or the hostile digestion process, as described previously [5]. At 9 to 14 days post-infection, the observed L. infantum Figure 1 L. longipalpis sandfly biting a Balb/C mouse. A single sandfly was confined within a vial and allowed to bite the animals entire ear. Note the blood engorgement of the sandfly (arrow). Inset: Dissected mouse ear showing the bite site as a small red dot (arrow). Figure 2 Parasite numbers in infected L. longipalpis (2 to 14 days after the infective bloodmeal). Flies were infected by membrane feeding on mouse blood containing 4 million logarithmic phase promastigotes/mL. Midguts were dissected on the indicated days post-feeding and scored for numbers of viable parasites. chagasi growth inside the sandfly indicated that a mature infection had been established reaching an average of 10,000 promastigotes/midgut (Figure 2). To develop a transmission model for laboratory use, we initially used two groups of animals: mice and hamsters. Twenty-eight ears of each animal group w (...truncated)