The transmission of Leishmania infantum chagasi by the bite of the Lutzomyia longipalpis to two different vertebrates
© Secundino et al; licensee BioMed Central Ltd. 2012
Received: 29 November 2011
Accepted: 19 January 2012
Published: 19 January 2012
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.
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.
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.
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 .
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 . 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 .
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 . 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 4×106/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.
Exposed-bite animal ears were processed for DNA extraction and real-time PCR as described elsewhere . 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.
In the New World, particularly in Brazil, leishmaniasis has followed the massive migration of people from rural areas into urbanized regions. In addition, the density of metropolitan areas and ease of travel between the two areas has led to increasing incidence rates of this vector-transmitted infectious disease . Studies with the Brazilian vector species are, thus, necessary to understand the underlying mechanisms of parasites, vector, and the vertebrate hosts, as well as the complex interactions between the three, which support disease onset and persistence.
In order to establish, for the first time, a transmission model of laboratory infection of visceral leishmaniasis, we used L. longipalpis infected with L. infantum chagasi to initiate infection by bite in laboratory animals. This approach probably is superior to the traditional needle inoculation method, which eliminates the natural transmission features (saliva, natural doses expelled through the proboscides).
Initially, we evaluated and compared hamsters and Balb/C mice as mammalian hosts for experimental infection. We concluded that there is no significant difference between these two animals in their utility as a model for transmission of visceral leishmaniasis by the vector bite. The two animals were bitten equally and the parasite numbers expelled into the ears by the L. longipalpis sandfly were similar, demonstrating that they are effective mammalian hosts for transmission of laboratorial infection.
Our results showed that the amount of L. infantum chagasi promastigotes delivered by a single L. longipalpis to the ears ranged from 4 to 10, 000 (mean: 1, 002). Coincidently, these numbers are similar to the findings from the force-feeding study  and others, in which L. longipalpis were artificially infected with L. mexicana and then forced to expel parasites into culture medium .
Interestingly, it is worth noting that in an experimental study of Old World cutaneous leishmaniasis the amount of L. major injected into mouse skin by a single sandfly, P. duboscqui could reach 100,000 parasites, but the majority of sandflies transmitted ~600 parasites .
Collectively, with our analysis, these findings indicated that the New World sandfly L. longipalpis cannot transmit as many parasites as the Old World sandfly. The majority of parasite loads transmitted by L. longipalpis are very low, since 75% of them transmitted < 300 parasites in the laboratory conditions. Only a few flies transmitted around 10, 000 parasites, a number that is still 10-fold less than that of the P. duboscqui. Further studies using other vector sandfly species are necessary to better understand this difference.
This study provided a strategy for initiating infection of experimental animals by vector bite that will facilitate future investigations into the complex and dynamic mechanisms of visceral leishmaniasis. The use of this model of parasite transmission may help to develop new strategies for prevention and treatment of leishmaniasis, a human life-treating disease.
This work was supported by grants from Fundação de Apoio a Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Tecnológico (CNPq), and Fundação Oswaldo Cruz (FIOCRUZ). We would also like to thank the FAPEMIG for its financial support of Vanessa C. de Freitas through a Junior Post-Doctoral Fellowship (CBB 00260/09).
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