Amblyomma sculptum source and maintenance

A. sculptum ticks from the Lab. de Doenças Parasitárias colony at the Federal Rural University of Rio de Janeiro (UFRRJ) were maintained on male rabbits (Orictolagus cuniculus, aged between 60 and 90 days and weighing approximately 2 Kg, provided by Veterinary Institute Cuniculture, UFRRJ), by a modification of a previously-described method [36]. A small area of the dorsal region was shaved, and bags of cotton cloth were glued to the rabbit’s skin. Within these bags were placed 20 pairs of adult male and female A. sculptum. To prevent removal of the bags by the rabbits, plastic cervical collars were placed on the necks of the animals as previously described [37].

Mycobacterium leprae preparation

Mycobacterium leprae Thai-53 strain [38], originally donated by Dr. James Krahenbuhl (National Hansen’s Disease Program, Laboratory Research Branch, Louisiana State University, Baton Rouge, LA, USA) was used to infect athymic nude mice bred in the Lauro de Souza Lima Institute (Baurú, São Paulo, Brazil) under germ-free conditions with 10⁴ bacilli in each posterior footpad. After six months the mice were sacrificed by a lethal dose of ketamine (300 mg/Kg) associated with xylazine (30 mg/Kg) (Syntec, São Paulo, Brazil), skin and bones were removed from the footpads aseptically in RPMI medium, and the remaining tissues were cut into small pieces with sterilized scissors and tweezers. The tissues were then gently dissociated for 2 h at 33°C in a solution comprising 15 mg/mL collagenase type I, 7 mg/mL dispase, 50 μg/mL DNAse and 100 μg/mL ampicillin (Life Technologies, Carlsbad, CA, USA) dissolved in Milli-Q (Millipore, Darmstadt, Germany) ultrapure water. After digestion, the resultant suspension was transferred to 1.5 mL tubes, centrifuged at 12,000 x g for 5 min and the supernatant was discarded. The pellet was washed twice with 1.5 mL water at 12,000 x g for 5 min, resuspended in 0.1 N sodium hydroxide (Sigma, St. Louis, MI, USA) and immediately centrifuged at 12,000 x g for 5 min. The purified bacilli were washed twice with 1.5 mL RPMI medium (LGC, Cotia, SP, Brazil) and the pellet was mechanically dissociated by passing the suspension through a 26 gauge needle. M. leprae preparations supplemented with 10% fetal bovine serum (FBS) (Cripion, Andradina, SP, Brazil) were stored for up to 3 days at 4°C. For the transformation protocol, M. leprae isolated from a skin lesion of a multibacillary leprosy patient from Igarapé Açú, Pará, northern Brazil, was used. This strain, designated PA4, had been cultivated in nude mouse footpads for six months and genotyped as a 4N strain.

The viability of both M. leprae strains was determined by assessing membrane integrity using the LIVE/DEAD BacLight bacterial viability kit (Life Technologies, CA, USA), performed according to the manufacturer’s instructions [39]. The percentage of viable bacilli in the preparation were estimated by counting the numbers of living bacteria (Cyto9 green signal excited at 488nm) and dead bacteria (propidium iodide red signal excited at 470nm), using an Axio Observer Z1 Zeiss microscope, with a HXP light source. Twenty fields each presenting at least 50 bacteria were quantified per preparation. Only bacterial preparations with viability of at least 85% as determined by the LIVE/DEAD method were used in the present work. Sterility of M. leprae preparations was checked by inoculation onto BHI and 7H9 liquid media (BioCen, Campinas, SP, Brazil), and Lowenstein and blood agar plates (BioCen, Campinas, SP, Brazil), which were monitored daily for 72 h (BHI and blood agar) and 45 days (7H9 and Lowenstein) at 37°C in order to exclude the presence of other contaminating microorganisms, including mycobacteria.

Artificial tick feeding and infection

Nearly-engorged adult female A. sculptum were detached from rabbits and weighed before being artificially fed, using plastic pipette tips [40,41] as follows. The ticks were divided into two groups; one group received at least 400μL of citrated rabbit blood containing 10⁷/mL viable M. leprae Thai-53 strain, and the other group received the same volume of uninfected blood. The tick mouthparts were introduced into the tips at an angle of approximately 30° and the ticks were maintained at 37±1°C with relative humidity of 80% for 24 h, when they were weighed again and ticks that had not gained at least 0.2 g were discarded. Engorged ticks were maintained at 80% humidity and 27°C for between 2 h and 15 days prior to dissection or for oviposition. Artificially-fed ticks were dissected and tissue samples were collected individually and stored in TRIzol (Thermo Fisher Scientific, MA, USA) or 10% buffered formalin; midguts were collected after 2 h and 15 days, while ovaries were collected after 2 days. Pools of 0.2 g eggs laid by the artificially-fed ticks were collected on the second and third days after the blood meal and homogenized in TRIzol. Larvae were obtained from the same amounts of eggs after 30 days’ incubation in sterile syringes [42] at 27°C with relative humidity approximately 80%. Two days after hatching, larvae were homogenized in TRIzol to investigate presence of M. leprae DNA and RNA.

Analysis of the potential of blood feeding larval ticks to transmit M. leprae

Two days after hatching, larvae originating from 0.2g of eggs laid by artificially-infected female A. sculptum were fed on a rabbit as described above for five days, the time required for this developmental stage to complete engorgement, and then removed. Subsequently, biopsies (5 mm punch) were performed in areas of the rabbit’s skin where there was evidence of previous insertion of the larval mouthparts. The biopsies were divided into two parts using a sterile scalpel; one part was extracted in TRIzol for quantitative PCR (qPCR) analysis of bacillary viability as described below, and the other part was fixed in 10% buffered formalin to perform immunolocalization of M. leprae as described below.

Analysis of M. leprae viability by qPCR

RNA and DNA were simultaneously extracted from test and control samples using TRIzol through a modification of the previously-described single-tube RNA extraction protocol [43]. Briefly, TRIzol was added to all samples to give a total volume of 1 ml. This solution was transferred to FastRNA tubes (FastRNA kit-Blue; MP Biomedicals, Santa Ana, CA). Samples were homogenized twice in the FastPrep FP 24 instrument (MP Biomedicals) at a speed setting of 6.5 for 45 s. Tubes were allowed to cool for 2 min between each round of homogenization. After homogenization, tubes were chilled on ice for 5 min, 200 μl chloroform-isoamyl alcohol (24:1) was added, and tubes were vortexed for 10 s and then centrifuged at 700 × g at 4°C for 5 min. The supernatant was transferred to another tube and centrifuged again at 12,000 × g for 10 min at 4°C. RNA was purified from 400 μl of the aqueous phase, any contaminating DNA was removed from the RNA preparations using the DNA-free kit (Ambion, Inc., Austin, TX) following the manufacturer’s instructions, the RNA was precipitated with isopropanol, washed with 70% ethanol, finally resuspended in 30 μl diethyl pyrocarbonate-treated H₂O, and stored at -80°C until use [44]. DNA was purified from the remaining aqueous phase and interphase of the FastRNA tubes. Briefly, 100 μl of 10 mMTris-EDTA (pH 8.0) and 150 μl chloroform-isoamyl alcohol (24:1) were added to the remaining aqueous phase and interphase (500 μl) and homogenized in the FastPrep FP 24 instrument twice. After centrifugation at 12,000 × g for 10 min, the aqueous phase was transferred into another tube and precipitated with 0.1 volume of 3 M sodium acetate pH 5.2 and two volumes of cold ethanol. The DNA pellet was washed in 70% ethanol, dissolved in 30 μl of sterile distilled water, and stored at -80°C until use. Qualitative RNA analysis was performed by agarose gel electrophoresis and quantification of both DNA and RNA was performed in a NanoDrop One apparatus (Thermo Fisher Scientific, MA, USA). The RNA was reverse-transcribed using random primers and Superscript III (Invitrogen, CA, USA) following the manufacturer’s instructions.

The levels of M. leprae 16S rRNA (as inferred from the resultant cDNA) and genomic DNA were determined in all tissues by qPCR, using as primer pair: sense 5’ GCA TGT CTT GTG GTG GAA AGC ‘3 and anti-sense 5’ CAC CCC ACC AAC AAG CTG AT ‘3, as described elsewhere [44]. The PCR protocol used was 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 min, monitoring SYBR Green fluorescence in an ABI StepOne Plus System (Applied Biosystems, CA, USA). Viability of M. leprae was determined by calculating the qPCR 16S cDNA:DNA ratio, according to the formula 2^-(ΔCT), where ΔCT = M. leprae 16S cDNA CT–M. leprae 16S DNA CT [44,45]. In order to convert 2^-(ΔCT) qPCR signals into numbers of live M. leprae genomes, we generated standard curves by adding different concentrations of living M. leprae, ranging from 10⁸ to 10³, into uninfected rabbit skin biopsies, tick cell cultures and tissues: midguts dissected 2h and 15 days after the blood meal, pools of 0.2 g eggs harvested between 48h and 72h after the blood meal, and larvae originating from 0.2g of eggs harvested 48h after hatching. We did not include ovarian tissue in this analysis due to difficulties in assuring absence of contamination from midgut contents during dissection. The angular coefficient of each of the five standard curves was used to determine the number of viable M. leprae in each condition. Control tissues and samples without the addition of DNA were negative for both cDNA and DNA targets.

Immunolocalization of M. leprae by fluorescence microscopy

As tick tissues are not well preserved by freezing protocols, M. leprae immunolocalization in tick tissues and rabbit skin biopsies was performed by fixation in 10% buffered formalin followed by standard paraffin embedding and sectioning as previously described [46]. The antigenic recovery was performed as follows: paraffin sections were dewaxed in xylol, hydrated in graded ethanol and distilled water, and heated in 10 mM sodium citrate buffer (pH 6.0) for 10 min in a 700 W microwave oven followed by 30 min to cool down to room temperature. M. leprae immunolocalization was performed using the monoclonal anti-lipoarabinomannan (LAM) antibody CS-35 reactive with Mycobacterium spp., kindly provided by the Biodefense and Emerging Infections Research Resources Repository: http://www.beiresources.org/TBVTRMResearchMaterials/tabid/1431/Default.aspx. Briefly, slides were permeabilized and blocked by 30 min incubation with 0.01% Triton X-100 and 10% FBS (Gibco/Life Technologies) in PBS pH 7.2 (permeabilization buffer). Then tissues were incubated for 2 h with the mouse IgG anti-LAM antibody CS-35 (diluted 1:50 vol/vol in permeabilization buffer) and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI Sigma-Aldrich, St. Louis, USA). Secondary antibodies, IgG anti-mouse conjugated with Alexa 594 (Invitrogen, CA, USA) diluted 1:250 in permeabilization buffer, were incubated with the samples for an additional 2 h and the tissues were then mounted in Vectashield (Vector Laboratories, CA, USA) and observed using a Zeiss Axio Observer Z1 microscope with a Colibri illumination system using a 590 nm LED with a Zeiss fluorescence filter 50 (Carl Zeiss, Heidenheim, Germany).

M. leprae infection of tick and human cell lines

Tick cell lines AVL/CTVM17 derived from larval Amblyomma variegatum [47], HAE/CTVM8 derived from embryonic Hyalomma anatolicum [48] and IDE8 derived from embryonic Ixodes scapularis [49] were maintained at Lab. de Doenças Parasitárias, Institute of Veterinary Medicine (UFRRJ) as described previously [50]. All cell lines were seeded in 24-well culture plates at a density of 2x10⁵ cells / well in 1 mL of complete culture medium as follows. HAE/CTVM8 cells were cultivated in L-15/H-Lac medium comprising equal volumes of Leibovitz’ L-15 medium (Gibco/Life Technologies) supplemented with 20% FBS (Cripion, Andradina, SP, Brazil), 10% tryptose phosphate broth (TPB, Sigma-Aldrich, Missouri, USA) and 2mM L-glutamine (Sigma-Aldrich, Missouri, USA) (L-15), and Hanks’ balanced salt solution (HBSS, Sigma-Aldrich, Missouri, USA) supplemented with 20% FBS, 0.5% lactalbumin hydrolysate (Sigma-Aldrich, Missouri, USA) and 2mM L-glutamine (H-Lac). IDE8 cells were grown in L-15B medium [51] supplemented with 10% FBS, 10% TPB, 0.1% bovine lipoprotein-cholesterol concentrate (MP Biomedicals, UK) and 2 mM L-glutamine (L-15B). AVL/CTVM17 cells were cultivated in medium comprising equal volumes of L-15, H-Lac and L-15B. Cells destined for microscopical analysis were seeded on coverslips, and all tick cells were maintained at 30°C. Human monocyte THP-1 cells were obtained from the American Type Culture Collection and maintained in RPMI 1640 medium (LGC Bioscience, Cotia, SP) supplemented with 10% FBS (Cultilab, Campinas, SP), without antibiotics. THP-1 cultures were grown at 37°C and transferred to 30°C when infected, within a humidified 5% CO₂ atmosphere. Cells were subjected to infection with M. leprae Thai-53 strain at a multiplicity of infection of 50 bacteria per cell (MOI 50:1) at 30°C. To examine association of M. leprae with tick cells, a total of 10⁷ freshly harvested M. leprae were stained with PKH67 dye (Sigma-Aldrich, Missouri, USA) following the manufacturer’s instructions with minor adaptations. Briefly 100μL of diluent provided with the kit and 1μL of PKH67 were added to the bacterial suspension. After 10 min incubation at room temperature with periodic mixing, 1mL of FBS was added to the bacterial suspension for 1 min to stop the reaction. The suspension was centrifuged at 14,000 x g for 5 min and the bacteria were resuspended in 100 μL of complete L15-B medium and immediately added to cell cultures seeded as above. For microscopy, after 24 h incubation at 30°C, cells in each well were fixed with 500 μL of 4% paraformaldehyde for 20 min, followed by three washes with HBSS and nuclear staining with DAPI. The coverslips were removed from the wells and placed on slides for analysis with a Zeiss Axio Observer Z1 fluorescence microscope as above. Samples were excited using 360 nm and 470 nm LEDs for respectively DAPI and PKH67, together with a Zeiss fluorescence filter 61. Images were acquired with a HMR Axiocam monochromatic camera controlled by Axiovision software version 3.2 (Carl Zeiss, Heidenheim, Germany), in which colors were arbitrarily attributed to the different channels. For flow cytometry, cells were gently washed with HBSS in order to remove any free bacilli, detached and fixed by the addition of 1% paraformaldehyde at 4°C until analysis using a BD Accuri C6 cytometer (BD Biosciences, California, USA) with the FL1-A channel set for counting 10,000 events. In both techniques, microscopy and flow cytometry, the index of bacterial association was expressed as the percentage of fluorescent cells.

In order to determine M. leprae growth inside tick cells, 2x10⁵ cells of each of the three cell lines were seeded in wells of a 24-well cell culture plate and infected 24 h later with live M. leprae Thai-53 strain, at a MOI of 50:1. After 24 h, 10 days and 20 days of infection, cultures were washed twice with sterile PBS and the cells were resuspended. The cell suspensions were centrifuged at 12,000 × g for 5 min and resuspended in milk-formaldehyde, which was prepared by centrifuging 1.5 mL of skimmed milk (Nestlé, Vaud, Switzerland) for 15 min at 12,000 × g, and transferring 1 mL of the supernatant to another tube containing 150 μL of formaldehyde (Sigma-Aldrich, Missouri, USA) and 9 mL of ultra-pure water. The pellets were resuspended at a dilution of 1:10 v/v in milk-formaldehyde and 10 μL aliquots were each spread over a 10 mm diameter circle etched on a glass slide (Electron Microscopy Sciences, Hatfield, USA). The glass slides were stained using the Kinyoun method for microscopic analysis [52]. Twenty fields with at least 50 bacteria were quantified per cell culture. The number of bacilli was estimated based on the ratio of the area of the circle on the slide area to the area of the field of view of the microscope obtained with a Zeiss Objective Plan-Apochromat microscope 100x/1.40 Oil objective (Carl Zeiss, Heidenheim, Germany). To calculate this ratio, the radius (r) of each area was measured using a 1mm microscope micrometer calibration slide ruler with 100 divisions (Cnscope, Beijing, China), and the areas were calculated using the formula π x r². Thus, the area of the circle on the slide and the area of the microscope field were determined as 78,539 mm² and 38 mm² respectively. This ratio (2.07x10³) was employed in the following equation: when A = average number of bacilli in ten microscope fields, B = dilution correction and 100 = volume correction from 10 μL to 1 mL.

M. leprae viability inside cells was determined by qPCR using 16S rRNA as target as described above. Briefly, 2x10⁵ cells of each of the three tick cell lines and the human monocyte THP-1 cells were seeded in 24-well cell culture plates. The differentiation of monocytes to macrophages was achieved by adding 200 ng/ml PMA (Sigma-Aldrich, Missouri, USA) to the medium. After 24 h, undifferentiated monocytes and dead cells were washed out with PBS, medium was resplaced, and another 24 h incubation preceded the infection with live M. leprae Thai-53 strain at a MOI of 50:1. After 20 days of infection, cultures were washed twice with sterile PBS and intracellular M. leprae DNA and RNA were extracted by adding TRIzol reagent.

Shepard`s model test of viability of tick cell-derived M. leprae

An IDE8 cell culture was infected with M. leprae Thai-53 strain at a MOI of 50:1 at 30°C. Culture medium was changed by removal and replacement of two-thirds of the total volume at weekly intervals. After 20 days of incubation, cells were washed and homogenized in 0.05% Tween 80 in sterile water. M. leprae was pelleted by centrifugation at 12,000 x g for 10 min, resuspended in 100 μL of PBS and counted as described above. Fifteen BALB/c mice were divided into three groups of 5 animals. Two groups were inoculated with 20 μL per foot pad of M. leprae suspension containing 10⁴ bacilli harvested from the IDE8 culture in a conventional Shepard's infection model [53]. A negative control was generated by treating animals in one of the two groups that received IDE8-derived M. leprae with 10mg/kg/week rifampicin by gavage. As a positive control, the third group were inoculated with the same number of M. leprae freshly purified from nude mouse foot pads. After six months, the mice were sacrificed by an intraperitoneal overdose of ketamine (300mg/kg) and xylazine (30mg/kg) and their foot pads were excised and macerated for bacillary counting after Kinyoun staining as above.

M. leprae transformation

An aliquot of 10⁹ bacteria of the M. leprae strain PA4 was subjected to transformation by electroporation with 5μg of pCHERRY3 plasmid DNA, kindly provided by Dr Suzie Hingley-Wilson of the University of Surrey, UK. This non-integrative plasmid, with a size of 6.3 kb, harbors a hygromycin resistance gene, a mycobacterial replication cassette, and the far-red fluorescence gene mCherry with expression driven by the Mycobacterium smegmatis promoter Psmyc [54]. The BioRad Micropulser TM (Hercules, CA, USA) electroporator parameters were adjusted for tension of 2.5 kV and electric resistance of 1000 Ω. The bacilli were pelleted by centrifuging for 10 min at 12,000 × g. The supernatant was discarded, and the pellet was dissolved in 200 μL sterile MilliQ water with 0.05% Tween 80. After adding the plasmid, the mixture was homogenized and transferred to a sterile electroporation cuvette at room temperature. After applying the electric pulse, transformed M. leprae were recovered with a sterile Pasteur pipette and immediately transferred to a 25 cm² culture flask containing 10⁷ IDE8 cells, maintained with weekly medium changes as above. After one week, selection of transformed M. leprae was performed by adding 50 μg/mL of hygromycin. After 1 week, 2 weeks and 2 months, a small number of cells were gently detached from the culture flask by shaking, and 10⁵ cells were placed in a glass bottomed 1.9 cm² cell culture dish (CELLview, Greiner Bio-One). mCherry expression by M. leprae was monitored using the Zeiss Axio Observer Z1 microscope as above, using a 590 nm LED with a Zeiss fluorescence filter 61 (Carl Zeiss, Heidenheim, Germany). Time-lapse photographic analysis of M. leprae-mCherry was performed 2 weeks after transformation, using 10⁵ cells placed in a glass bottomed CELLview culture dish and the same microscope with the Zeiss Definitive Focus (Carl Zeiss, Heidenheim, Germany) accessory with stage temperature stabilized at 30°C. The medium was supplemented with 150 μg / mL ofloxacin (Sigma, St. Louis, MI, USA), and 5 h later the video was started, with acquisition of one frame every 5 min over a period of 3 h.

Genotyping by multiple Locus Variable number analysis (MVLA)

DNA extracts from M. leprae-infected tick cell cultures were submitted to genotyping by MLVA analysis of 16 variable-number tandem-repeat (VNTR) loci, as previously described [55]. For each sample, four multiplex PCR reactions were performed that generated 16 amplicons and allele copy number was determined by denaturation of amplicons and capillary gel electrophoresis on the sequencer ABI 3130 Genetic Analyzer, using the internal molecular weight sizing standards LIZ 500 (Thermo Fisher Scientific, MA, USA) and copy number definition with the Peak Scanner software (Applied Biosystems, MA, USA).

Statistical analysis

All numerical data were analyzed using nonparametric tests, with Dunn’s multiple comparisons test to compare relevant groups, or Mann Whitney test to compare continuous variables. All statistical analyses were performed with GraphPad Prism software.

Ethics statement

The use of rabbits in this study was supported by the authorization granted by the Committee on Ethics in the Use of Animals of UFRRJ (CEUA-UFRRJ) (Rio de Janeiro, Brazil) under No. 380/2013, process 23083.006255 / 2013–25. The use of mice was approved in license number 219/11 by the Animal Welfare Committee of Sagrado Coração University (CEUA-USC) (São Paulo, Brazil), responsible for animal care and inspections at the Instituto Lauro de Souza Lima where all mouse experiments were performed. All procedures were performed in accordance with the Brazilian guide for the production, maintenance and use of animals in teaching or scientific research activities from CONCEA (Conselho Nacional de Controle de Experimentação Animal). In the present work we used the M. leprae strain PA4 isolated from a skin lesion of an anonymous relapsed multibacillary patient from Igarapé Açú, Pará, Brazil, maintained in nude mice in the M. leprae Biobank at Lauro de Souza Lima since 2008. The Lauro de Souza Lima Research Ethics Committee (ILSL-CEP) was notified and permitted the use of this sample.