Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites

Abstract

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

Vector permissiveness, vector competence, and vector incrimination are different designations that qualify vectors [1, 2]. The first is the ability of the vector to sustain an infection, which is encompassed by the second, that is the capacity for acquisition, maintenance, and transmission of a given pathogen, which in turn is converted to the third when these abilities are verified in natural conditions.

In sand fly vectors, responsible for the transmission of more than 20 Leishmania species pathogenic to humans [3], the dichotomy of specific versus permissive vectors has been recognized for a long time [4]. Lutzomyia longipalpis sand flies represent the archetype of a permissive Leishmania vector under laboratory conditions. Although they are incriminated only as a Leishmania infantum vector in nature and are the most relevant transmitting agent of visceral leishmaniasis in the New World, laboratory colonies have been reproducibly demonstrated to sustain infections with different Leishmania species such as L. major, L. donovani, L. mexicana, and L. amazonensis [5–8]. However, no study had completely determined if this permissive potential for infection would translate to vector competence, as this may not always be the case considering the number of factors that may influence vector transmission [9].

Here we evaluated the capacity of Lu. longipalpis sand flies to acquire, maintain, and transmit L. major parasites by reproducing the natural Leishmania transmission cycle under laboratory conditions.

METHODS

Ethics Statement

All animal experiments were carried out in accordance with the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee under the animal protocol LMVR4E.

Mice

Six-week-old female BALB/c mice were obtained from Charles River laboratories and housed under pathogen-free conditions at the NIAID Twinbrook animal facility (Rockville, MD) with water and food ad libitum.

Parasites and Needle Infection

A cloned line of Leishmania major (WR 2885) was used [10]. The WR2885 strain was isolated 29 August 2008 from a lesion on the right upper arm of a US soldier. It was acquired in Iraq and the cloned line was identified as L. major by polymerase chain reaction (PCR) and by isozyme assessment by a College of American Pathologists certified laboratory. Promastigotes were maintained at 26°C in Schneider’s insect medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (all Thermo Fischer Scientific). Metacyclic promastigotes, purified as previously described [11], were used to infect mouse footpads or ears (intradermal injection of 10⁶ or 10³ parasites, respectively, in 10 µL phosphate-buffered saline [PBS]).

Sand Flies, Natural Infection, and Follow-up

Lu. longipalpis sand flies were mass reared at the Laboratory of Malaria and Vector Research insectary as previously described [12]. Adult females were maintained on a 30% sucrose diet and were starved for 12 hours before feeding. Sand flies, 5–7 days old, were allowed to feed on the active lesions (either on footpads or ears) of BALB/c mice infected with L. major, as previously reported. Briefly, either (1) 20 sand flies were applied to each infected mouse ear (6 weeks post infection) using vials with a meshed surface held in place by custom-made clamps [5]; or (2) sand flies in mesh-covered containers were allowed to feed on infected mice footpads (3 weeks post infection) [10]. After infection, blood-fed females were sorted and kept on a 30% sucrose diet. At 7 and 12 days post infection sand flies were collected to assess the infection status.

Sand Fly Infection Status Evaluation

Under a stereomicroscope, sand fly midguts were dissected in PBS and transferred to individual microtubes (Denville Scientific) with 50 μL of formalin solution (0.005% in PBS). Midguts were homogenized and 10 μL loaded onto disposable Neubauer chambers (Incyto). Slides were observed under a phase contrast microscope (Zeiss) at 400× magnification. The frequency of infection, the total parasite numbers, and the metacyclic frequency [6] were determined.

Transmission of L. Major via Sand Fly Bites

Only Lu. longipalpis females that fed on BALB/c infected footpads were used. At day 12 post feeding, 10 sand flies were applied to each mouse ear, using vials with a meshed surface held in place by custom-made clamps, and allowed to feed. The number of blood-fed flies was determined by observing them under a stereomicroscope.

Posttransmission Follow-up and Experimental Endpoints

Animals were monitored on a weekly basis to follow the development of lesions caused by L. major infection by measurements of ear thickness with a Digimatic caliper (Mitutoyo Corp.). Animals were euthanized at 3 weeks post infection for parasite burden determination.

Parasite Burden Determination

Euthanasia was performed by cervical dislocation under isoflurane anesthesia in all cases (Piramal Healthcare). Ears were collected, immersed in 70% ethanol and then in PBS. Ear cell suspensions were obtained as previously described [5]. Ear parasite burdens were assessed by the limit dilution method. Briefly, ear cell suspensions were serially diluted in 96-well plates containing 50 µL biphasic medium (Novy-MacNeal-Nicolle medium with 10% defibrinated rabbit blood overlaid with 200 µL Schneider’s insect medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin; all Thermo Fischer Scientific), and incubated for 14 days at 26°C. The total number of parasites per ear was calculated as previously [5].

Data Representation and Statistics

Results obtained in at least 2 independent experiments are shown per individual sand fly/mouse, with a representation of the group mean value ± standard deviation. Statistical analysis was performed using GraphPad Prism software v6.01. The nonparametric Mann-Whitney test was used to access statistical differences, with at least P ≤ .05.

RESULTS

To understand if the vector permissiveness of Lu. longipalpis would translate to vector competence for L. major parasites, we reproduced the natural Leishmania transmission cycle under laboratory conditions. We allowed Lu. longipalpis to feed from active L. major lesions in BALB/c mice footpads or ears, selected the engorged flies, and evaluated their infection outcome at 2 time points, as well as their capacity to transmit parasites to a new host and cause disease, thereby closing the infection cycle (Supplementary Figure 1).

From the sand flies who took a blood meal from active footpad cutaneous leishmaniasis lesions, more than 39% were infected at both 7 and 12 days post feeding (Figure 1A). Remarkably, within the infected sand flies, we observed a healthy and sustained parasite population that amplified with time, from around 18 000 parasites/midgut at day 7, to more than 90 000/midgut at day 12 post feeding (Figure 1B). We also observed an infection maturation, that is an increase in the frequency of metacyclic L. major parasites from less than 15% at day 7 to more than 50% at day 12 post feeding (Figure 1C). Similarly, when Lu. longipalpis sand flies fed on an active ear lesions caused by L. major infection, we obtained comparable results. From the flies that took a blood meal, more than 30% were infected both at 7 and 12 days post feeding (Supplementary Figure 2A). Additionally, an average of around 10 000 parasites/midgut was determined at day 7 post feeding, which doubled by day 12 post blood meal (Supplementary Figure 2B). Furthermore, the metacyclic parasite frequency increased from day 7 to day 12 post feeding, although in a less expressive manner (Supplementary Figure 2C).

Figure 1.

Infection outcome of Lutzomyia longipalpis sand flies blood fed on active Leishmania major lesions developed in BALB/c mice footpads. Sand flies were allowed to feed on active footpad lesions of BALB/c mice infected with L. major (3 weeks post infection). Blood-fed females were sorted, fed a 30% sucrose diet, and dissected 7 and 12 days post feeding for infection status assessment: A, frequency of infection; B, total parasite numbers per infected sand fly; C, metacyclic population frequency per infected sand fly. Results from 2 independent experiments are shown. B and C, Each symbol represents 1 sand fly. Frequencies or average and SD of the values within each group are shown. Statistical differences are by nonparametric Mann-Whitney test. * P ≤ .05, **** P ≤ .0001.

We then tested if these sand flies could transmit Leishmania parasites and cause disease in naive animals. We exposed naïve mouse ears to 10 sand flies and from those a median number of 4.5 blood fed. From 15 naive BALB/c mice that were exposed in the ear to Lu. longipalpis sand fly bites, 13 animals developed cutaneous leishmaniasis ear lesions, as early as 2 weeks post exposure (Figure 2A and 2B). The lesion development was heterogeneous (Figure 2A) with some animals showing, at 3 weeks post transmission, small localized single lesions, while others showed larger lesions with scarring (Figure 2B). The parasite burden assay detected parasites in 14 of 15 exposed ears, with an average parasitemia of 5 million parasites/ear, which demonstrated the success of transmission and the closure of the infection cycle (Figure 2C).

Figure 2.

Lutzomyia longipalpis sand flies naturally infected with Leishmania major parasites are able to transmit cutaneous leishmaniasis to a new naive host. Sand flies were allowed to feed on active footpad lesions of BALB/c mice infected with L. major (3 weeks post infection). Blood-fed females were sorted, fed a 30% sucrose diet, and used 12 days post feeding in tentative transmission experiments to naive BALB/c mice ears (10 sand flies per ear were allowed to feed). Lesion pathology was evaluated on a weekly basis up to week 3 post transmission, when the animals were euthanized for parasite burden quantification. A, Ear thickness measurements per individual animal. B, Illustrative images of ear lesions at week 3 post transmission. C, Total ear parasite burdens at the 3-week time point. Results from 2 independent experiments are shown and each symbol represents 1 animal with the average and SD.

DISCUSSION

In this study we explored the vector competence of Lu. longipalpis to a Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, because not only could this sand fly species acquire the parasite through a blood meal taken from active murine cutaneous footpad lesions, but also the parasite was maintained in a sufficiently efficient manner that it was effectively transmitted to new hosts.

Remarkably, the infection status we report here for Lu. longipalpis sand flies is similar to that reported for Phlebotomus papatasi sand flies [10], the incriminated vector of L. major responsible for the transmission of cutaneous leishmaniasis in the Old World [3]. Serafim et al showed that, in a similar experimental context, 9 days post feeding P. papatasi sand flies carried an average of 6000 L. major parasites/midgut (compared to 18 000 we report for Lu. longipalpis sand flies at day 7 post feeding), a number that doubled by day 12 post feeding (compared to the 5-fold increase we report here) [10]. Additionally, although the mean metacyclic percentages recorded in the same experimental context by the abovementioned study [10] were around 13% and 23% at day 9 and 12 post infection, respectively (compared to the 10% and 50% we report here for days 7 and 12 post feeding), these calculations were performed considering the entire sand fly population, including the noninfected ones, which consequently contributed to an underestimation of the actual infection status. Although there was a difference in metacyclic percentage, in accord with the results reported here for Lu. longipalpis sand flies, the expected maturation of infection was observed from day 9 to 12 post feeding for P. papatasi sand flies also, determined by the increase in metacyclic parasite frequency/sand fly midguts [10].

One may argue that the abovementioned comparison of results, as well as the data we report here, may be irrelevant in the natural context. It is well known and we acknowledge that Lu. longipalpis sand fly distribution is restricted to the New World [7] while L. major parasites (and the reported cutaneous disease cases caused by them) are confined to the Old World [3], meaning that the probability of parasite-vector geographic overlap is quite low. However, such overlap is not impossible. Modern transportation has brought the world closer; the movement of people, animals, and goods has never been as fast as it is today. And if, in one direction, the accidental importation of Lu. longipalpis sand flies (as is reported for other vectors [13]) to L. major endemic areas seems unlikely, requiring either adaptation of the vector to completely new ecological settings or its arrival in areas ecologically similar, in the other direction, the importation of L. major parasites via a reservoir to Lu. longipalpis endemic territories is not a far-fetched hypothesis. In line with this possibility, we have to keep in mind it has been suggested that L. infantum parasites, which are transmitted naturally by Lu. longipalpis sand flies, were imported to the New World by travelers 500 years ago [3].

This discussion can be extended to other known permissive vector species and the real risk of emergence of leishmaniasis of atypical etiology in Europe. Recently, P. perniciosus and P. tobbi sand flies were shown to be permissive (or even competent) to different Leishmania tropica strains [14, 15]. The higher geographic potential for overlap of vector and parasite species, due to the existence of major migratory events in the region (mainly, but not exclusively, as a consequence of conflict) may therefore contribute to the short-term establishment of new leishmaniasis foci, an hypothesis that deserves a high priority for epidemiological attention [4, 15].

In summary, here we prove that Lu. longipalpis sand flies are competent vectors of L. major parasites. It is important to state, however, that we are not incriminating Lu. longipalpis sand flies as L. major vectors. However, our data gives relevance to the Lu. longipalpis-L. major pairing that can be used in the laboratory as a cutaneous leishmaniasis natural transmission model (if access to P. duboscqi or P. papatasi sand flies is restricted), which is always better than Leishmania needle inoculation in the context of both basic and translational research. Additionally, from our study, we hope to open avenues of opportunity to the exploitation, particularly but not only of Lu. longipalpis competence to other Leishmania species, especially those with a higher potential for vector-parasite geographic overlap.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Author contributions. P. C., A. C. A. M. P., and F. O. conceived, designed, and performed the experiments. P. C. and A. C. A. M. P. analyzed the data. J. G. V. assured the funding and contributed reagents, materials, and analysis tools. P. C. and F. O. wrote the original draft. All the authors critically discussed the results, and revised, edited, and approved the manuscript.

Acknowledgments. We are grateful to the National Institute of Allergy and Infectious Diseases Animal Facility for all the technical assistance provided.

Disclaimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial support. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health; P. C. was supported by Foundation for Science and Technology, Portugal (grant number SFRH/BD/121252/2016); and A. C. A. M. P. was supported by CNPq-Science without Borders Program, Brazil.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.