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Zoonotic Visceral leishmaniasis (ZVL) is a neglected tropical disease that in the Americas is caused by the infection of Leishmania infantum and the domestic dog (Canis familiaris) is the main parasite reservoir in urban areas. The parasite is mainly transmitted by populations of the sibling species Lutzomyia longipalpis that has been spreading in countries including Brazil, Argentina, Paraguay and more recently Uruguay. Although bionomic parameters such as population survival and the duration of the gonotrophic cycle are critical in evaluating vector capacity, field studies have rarely been applied to sand fly populations. The present study sought to evaluate basic bionomic parameters related to the vectorial capacity of the (S)-9-methylgermacrene-B population of the Lu. longipalpis complex in a visceral leishmaniasis area of Sao Paulo state. The daily survival rate, the duration of the gonotrophic cycle and the dispersal pattern were evaluated through the mark- release-recapture method. A total of 1,547 males and 401 females were marked and released in five experiments carried out between February 2013 and February 2014. The higher recapture rates occurred within 100 meters of the release point and the estimated daily survival rates varied between 0.69 and 0.89 for females and between 0.69 and 0.79 for males. The minimum duration of the gonotrophic cycle observed was five days. The absolute population size, calculated ranged from 900 to 4,857 females and from 2,882 to 9,543 males. Our results demonstrate a high survival rate of this vector population and low dispersal that could be associated with the presence of all necessary conditions for its establishment and maintenance in the peridomiciles of this area. Our findings contribute to the basic data necessary for the understanding of ZVL dynamics and the evaluation of the implementation of prevention and control measures.

Several parameters should be addressed before incriminating a vector for Leishmania transmission. Those may include its ability to become infected by the same Leishmania species found in humans, the degree of attractiveness for reservoirs and humans and capacity to sustain parasite infection under laboratory conditions. This study evaluated the vectorial capacity of Lutzomyia cruzi for Leishmania infantum and gathered information on its ability to harbor L. amazonensis. Laboratory-reared Lu. cruzi were infected experimentally by feeding them on dogs infected naturally with L. infantum and hamsters infected with L. amazonensis. Sand fly attractiveness to dogs and humans was determined using wild caught insects. The expected daily survival of infected Lu. cruzi, the duration of the gonotrophic cycle, and the extrinsic incubation period were also investigated for both parasites. Vector competence was investigated for both Leishmania species. The mean proportion of female sand flies that fed on hosts was 0.40. For L. infantum and L. amazonensis, Lu. cruzi had experimental infection rates of 10.55% and 41.56%, respectively. The extrinsic incubation period was 3 days for both Leishmania species, regardless of the host. Survival expectancy of females infected with L. infantum and L. amazonensis after completing the gonotrophic cycle was 1.32 and 0.43, respectively. There was no association between L. infantum infection and sand fly longevity, but L. amazonensis-infected flies had significantly greater survival probabilities. Furthermore, egg-laying was significantly detrimental to survival. Lu. cruzi was found to be highly attracted to both dogs and humans. After a bloodmeal on experimentally infected hosts, both parasites were able to survive and develop late-stage infections in Lu. cruzi. However, transmission was demonstrated only for L. amazonensis-infected sand flies. In conclusion, Lu. cruzi fulfilled several of the requirements of vectorial capacity for L. infantum transmission. Moreover, it was also permissive to L. amazonensis.

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted mainly by Lutzomyia longipalpis. However, canine cases have been reported in the absence of this species in the Greater São Paulo region, where Pintomyia fischeri and Migonemyia migonei are the predominant species. This raises the suspicion that they could be acting as vectors. Therefore, this study sought to investigate specific vector capacity parameters of these species and to compare them with those of Lu. longipalpis s.l. Among these parameters the blood feeding rate, the survival, and the susceptibility to the development of Le. infantum were evaluated for the three species, and the attractiveness of dogs to Pi. fischeri and Mg. migonei was evaluated. The estimated interval between blood meals was shorter for Lu. longipalpis s.l, followed by Pi. fischeri and Mg. migonei. The infection rate with Le. infantum flagellates in Lu. longipalpis was 9.8%, in Pi. fischeri 4.8%, and in Mg. migonei nil. The respective infective life expectancies (days) of Lu. longipalpis, Mg. migonei, and Pi. fischeri were 2.4, 1.94, and 1.68. Both Pi. fischeri and Mg. migonei were captured in the kennel with a predominance (95%) of Pi. fischeri. Considering the great attractiveness of dogs to Pi. fischeri, its susceptibility to infection by Le. infantum, infective life expectancies, and predominance in Greater São Paulo, this study presents evidence of Pi. fischeri as a potential vector of this parasite in the region., (© F. Galvis-Ovallos et al., published by EDP Sciences, 2017.)

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 microL and photomicrographed. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10(2) promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.