Sand Fly Colony

For all experimental infections, well-adapted laboratory colony of P. perniciosus (originating from Spain) was used. Colony was maintained in the insectary of the Charles University in Prague under standard conditions (26 ± 1°C with 60–70% relative humidity, a light: dark cycle of 14 h: 10 h and 50% sucrose solution). Details of the colony maintenance are described by Volf and Volfova (2011) and Lawyer et al. (2017). For experimental infection in IRD Montpellier, 500 P. perniciosus sand flies were shipped weekly (4 d before experiments). After reception, individuals were transferred in nylon mesh cages and maintained in climatic chamber under the same conditions as in CUNI.

Cultivation of Leishmania Parasites

Leishmania infantum mCherry (MHOM/TR/2000/OG-VL) promastigotes (transfected with red fluorescence protein) were cultivated in M199 medium (Sigma) containing 20% heat-inactivated fetal bovine serum (FBS) (Gibson), supplemented with 2% sterile urine, 1% Basal Medium Eagle vitamins (Sigma), 250 µg mL^–1 amikacin (Amikin, Bristol-Myers Squibb) and 150 µg mL^–1 selective antibiotic neomycin (Sigma). Each week parasites were passaged to the fresh medium, during all experiments, parasites with in-vitro passage number less than 10 were used. For experimental infection in IRD Montpellier, two vials containing frozen L. infantum mCherry (MHOM/TR/2000/OG-VL) were shipped on dry ice from CUNI, 15 d before the first experimental infection, allowing the IRD hosts to establish stable cultures of parasites.

Cultivation of Macrophages

Immortalized macrophage cell line J744 originating from BALB/c mice was used. J744 macrophages were cultured at 37°C with 5% CO₂ in complete RPMI-1640 medium (Sigma) containing 10% FBS, 1% penicillin–streptomycin (Sigma) and 0.05 mM of β-mercaptoethanol. Macrophages were seeded at about 2x10⁴ cells/mL in culture medium and were subcultured every 5 d. For experiments in IRD Montpellier, two vials containing frozen J744 macrophage cell lines were shipped on dry ice from CUNI, 15 d before the first experimental infection, allowing IRD to establish stable cultures of macrophages.

Infection of Macrophages with Leishmania Promastigotes and Their Transformation to Amastigotes

Macrophage infection was performed 3 d prior to the infection feeding of sand flies. Leishmania promastigotes in a stationary phase of growing were washed two to three times in sterile saline solution and counted in hemocytometer. Three- to four-day-old macrophages were counted in hemocytometer and later on exposed to stationary-phase parasites at a ratio of eight promastigotes per one macrophage. Infected macrophages were cultivated in the same medium mixture as noninfected ones.

Preparation for Promastigote-Initiated Infection

For promastigote-initiated infection, Leishmania promastigotes from log-phase cultures (4 d postinoculation) were resuspended in heat-inactivated rabbit blood at concentration of 10⁶ promastigotes per millilter.

Preparation of Parasites for Amastigote-Initiated Infection

For amastigote-initiated infections, Leishmania parasites were cocultivated with macrophage cell line J774 for 72 h. Noninternalized parasites were removed by washing with preheated culture medium. The macrophages were removed from the culture plates by extensive washing with cold saline solution, centrifuged at 300×g, 4°C for 10 min and resuspended in saline solution. Ten microliters of mixture were used for amastigotes per macrophage counting under fluorescent microscope. After counting, macrophages were resuspended in heat-inactivated rabbit blood for sand fly infections at the concentration of 10⁶ amastigotes per mlilliter.

Feeding Process, Postfeeding Manipulation and Dissection of Sand Flies

Process of sand fly feeding, postfeeding manipulation and dissection varied between BSL2 and BSL3 laboratories. Even though the preparation of parasites and its dosage for experimental infection was the same, process of feeding was performed according to the special requirements of the laboratories and their level of protection (BSL2 vs BSL3) ( Supp Table 1 [online only]).

Experimental Infections and Sand Fly Manipulation in BSL2 Facilities

Twenty-four hours prior to the infection, approximately 200 sand fly females (5–9 d old) were put in separate mesh cages and deprived of sugar. During the infection feeding, lower part of the glass feeder was placed directly inside the mesh cage through the sleeve, allowing sand fly direct access to the chicken skin membrane. Constant temperature of 37°C for heating the blood in the feeders was maintained by water bath with external circulation. Feeding was performed for 2 h at 26°C in a darkened room.

Twenty-four hours after blood feeding engorged females were separated using mouth aspirators into a separate mesh cage and kept under standard conditions. Briefly, the cage was placed in a plastic bag with moist cotton wool to maintain high humidity and kept at 26°C in a separate incubator. Day after blood feeding, small piece of cotton wool pad soaked by 50% sucrose solution was provided to females on Petri dish placed directly inside the cage.

For the dissection, females were aspirated from the mesh cages into a plastic pot and cooled on ice to anesthetize them. Guts of experimentally infected sand flies were dissected under a stereomicroscope in saline solution by fine tweezers and/or entomological pins. Females were dissected before defecation (early stage of infection at day 2), and after defecation (late stage of infection) at day 8 and 11 post-bloodmeal (PBM).

Abundance and localization of Leishmania parasites in the sand fly gut was examined by light and fluorescent microscopy. Parasite loads were graded as light (less than 100 parasites per gut), moderate/medium (100–1,000 parasites per gut), and heavy (more than 1,000 parasites per gut) (Myskova et al. 2015).

Experimental Infections and Sand Fly Manipulation in BSL3 Facilities

The use of mesh cages for sand fly infectious feeding within BSL2 facilities is a common practice in laboratories studying Leishmania experimental infections in sand flies. However, within BSL3 facilities, due to the work with more dangerous pathogens, it is necessary to reduce the risk of exposure and contamination. As opening the mesh cages and insertion/retrieval of feeders containing infectious material pose a significant risk of sand fly escape, for membrane feeding within BSL3 laboratory we specially designed cylindrical plastic containers with large opening (approximately 8 cm). Bottom of the container was closed with styrofoam cap while tight mesh was placed on the top (Picture 1). The net used for the purpose of feeding had a larger hole diameter (about 0.020 inches), compared with the standard nets used for sand fly maintenance cages (thickness 0.0078 ± 0.0015 inches), which allowed sand flies to feed though the mesh. During the infection, containers were set upright and feeders were placed on top of the mesh. Feeding of approximately 200 females per cage (5–9 d old) was performed inside the climatic chamber at a temperature of 26°C during 2 h. The blood in the feeders was maintained at constant temperature (37°C) by water bath with external circulation.

Picture 1.

Feeding pots—specially designed containers used during infection feeding of sand flies conducted in BSL3 facilities.

Despite the fact that engorged females are very sensitive and it is better not to handle them during the first 24 h postingestion, due to the safety regulations within BSL3 laboratory ( Supp Table 1 [online only]) leaving infected blood-stained mesh freely exposed is prohibited, thus females were separated immediately after ingestion. Since mandatory equipment in BSL3 facilities considers the use of protectant mask for mouth and nose, use of mouth aspirators for separation of blood fed females is not applicable. For this purpose, immediately after feeding, plastic containers were placed on ice to anesthetize sand flies. Separation of engorged females was done with soft tweezers in Petri dishes kept on ice. Engorged females were transferred into the cardboard containers with a maximum of 30 individuals per container. Containers were made out of tick cylindrical cardboard (diameter 10 cm, length of cylinder 10 cm) which was closed with tick mesh on both sides (Picture 2). Containers with sand flies were enveloped by plastic bag, together with wet cotton pad which was placed inside to maintain high humidity and put in incubator (26°C). Twenty-four hours after blood feeding wool pad soaked by 50% sucrose solution was placed on the top mesh of the container.

Statistical Analysis

Statistical analyses of amastigote- and promastigote-initiated infection (intensity of infection and localization) in different BSL were done by Fisher's exact test using R software version 3.1.0 (R Development Core Team 2018). The results obtained in CUNI and IRD were compared using Pearson's Chi-squared test using R software. A P-value of < 0.05 was considered significant. Statistical results were presented in legends of Figs 1 and 2.

Experimental Infections in BSL2 Facilities

Promastigote-initiated infection

In promastigote-initiated infection, a total of 169 blood fed sand fly females were dissected and examined for presence and localization of Leishmania parasites in the gut with a total infection rate of 82.8%.

Picture 2.

Resting pot—specially designed containers used for storing of blood fed females after infection feeding conducted in BSL3 facilities.

Fig. 1.

Infection rate and parasite loads: A – BSL2: B – BSL3. (PRO – promastigotes; AMA – amastigotes). Comparison of parasite loads among promastigote- and amastigote-initiated infection was evaluated by Fisher exact test on day 2 PBM (BSL2: P = 0.843, df = 3; BSL3: P = 0.164, df = 3), 8 PBM (BSL2: P = 0.6927, df = 3; BSL3: P = 0.088, df = 3) and day 11 PBM (BSL2: P = 0.3506, df = 3; BSL3: P = 0.004, df = 3). Differences between results obtained in CUNI and IRD were evaluated using Pearson's Chi-squared test (all days and all experiments together): P = 0.046, df = 3.

Infection rates and intensities of infection

On the day 2 PBM, 60 sand flies were dissected with 95% infection rate. High-intensity infections were detected in 56.6% of females, medium in 30%, and low infections in 8.3% (Fig. 1A). On day 8 PBM, 71 sand flies were dissected with infection rate of 83%. High intensity infections were detected in 56.3%, medium in 21.1%, and low infection in 5.6% of blood fed females. Finally, on day 11 PBM, 38 sand flies were dissected with infection rate of 63.1%. High, moderate, and low intensity infections were recorded in 55.2%, 2.6%, and 5.2% of dissected females, respectively (Fig. 1A).

Localization of parasites in sand fly gut

On day 2 PBM, all parasites were localized inside the endoperitrophic space surrounded by the peritrophic matrix (Fig. 2A). On day 8 PBM, colonization of the stomodeal valve was observed in 76.2% of infected females (Fig. 2A). Similarly, on day 11 PBM, parasites reached the stomodeal valve in 75% of infected females (Fig. 2A).

Fig. 2.

Localization of parasites in sand fly gut: A– BSL2, B – BSL3D. (BM – bloodmeal; AM – abdominal midgut;TM – thoracic midgut; AM – abdominal midgut). Comparison of parasite localization inside the sand fly gut among promastigote- and amastigote-initiated infection was evaluated by Fisher exact test on day 2 PBM (BSL2: p-value = 1, df = 2; BSL3: p-value = 1, df = 2), 8 PBM (BSL2: p-value = 1, df = 2; BSL3: p-value = 1, df = 2) and day 11 PBM (BSL2: p-value = 1, df = 2; BSL3: p-value = 1, df = 2). Differences between results obtained in CUNI and IRD were evaluated using Pearson's Chi-squared test (all days and all experiments together): P < 0.05, df = 5.

Experimental Infections in BSL3 Facilities

In BSL3 facilities, glass feeders were placed on the mesh on the top of the feeding containers as compared to the BSL2 facilities where the glass feeders were placed directly inside the mesh cages allowing sand flies direct access to the chick skin membrane. Presence of the mesh did not seem to affect the feeding rate since majority of females took a bloodmeal (≈70% in BSL3; ≈75% in BSL2).