Name: In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase (Video) | JoVE Journal
Uploaded: 2025-04-04T12:20:00.000+00:00
Duration: 5 min 55 s
Description: Learn bioluminescent imaging of Leishmania infantum infection in golden hamsters using gingival vein inoculation of axenic amastigotes. This method enables real-time tracking of parasite burden for preclinical research.

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Funda&#231;&#227;o de Apoio ao Desenvolvimento do Ensino, Ci&#234;ncia e Tecnologia do Estado de Mato Grosso do Sul- FUNDECT. PPSUS/ Decit-MS/CNPq/SES provided financial support for this research. Thanks to Monique Ribeiro de Lima for her advice about the routes of inoculation. This project was developed under the Cooperation Agreement No 258/2017 between FIOCRUZ, and Universidade Federal do Rio de Janeiro- UFRJ.&#160;The team is sincerely grateful to the video producers Ricardo Baptista Schmidt and Genilton Jos&#233; Vieira from the Science Popularization Center (IOC) for their invaluable support and assistance in filming the protocols and conducting the interviews.

<ol><li>Ruiz-Postigo, J. A., et al. Global leishmaniasis surveillance, 2022: assessing trends over the past 10 years. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRuiz-Postigo%2C+JA+%22WHO%2C+Weekly+epidemiological+record+n%C2%B0+40%22">WHO, Weekly epidemiological record n° 40</a>. , (2023).</li>
<li>Faraut-Gambarelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+and+in+vivo+resistance+of+Leishmania+infantum+to+meglumine+antimoniate%3A+a+study+of+37+strains+collected+from+patients+with+visceral+leishmaniasis%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis.</a> Antimicrob Agents Chemother. 41 (4), 827-830 (1997).</li>
<li>Ribeiro-Romão, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+evaluation+of+lesion+development%2C+tissue+damage%2C+and+cytokine+expression+in+golden+hamsters+%28Mesocricetus+auratus%29+infected+by+inocula+with+different+Leishmania+%28Viannia%29+braziliensis+concentrations%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Comparative evaluation of lesion development, tissue damage, and cytokine expression in golden hamsters (Mesocricetus auratus) infected by inocula with different Leishmania (Viannia) braziliensis concentrations.</a> Infect Immun. 82 (12), 5203-5213 (2014).</li>
<li>Gomes-Silva, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((hamster+%28Mesocricetus+auratus%29+as+an+experimental+model+for+Leishmania+%28Viannia%29+braziliensis+infection%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">hamster (Mesocricetus auratus) as an experimental model for Leishmania (Viannia) braziliensis infection.</a> Parasitology. 140 (6), 771-779 (2013).</li>
<li>Moreira, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((hematological+and+biochemical+alterations+in+hamster+%28Mesocricetus+auratus%29+experimentally+infected+with+Leishmania+infantum+through+different+routes+of+inoculation%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">hematological and biochemical alterations in hamster (Mesocricetus auratus) experimentally infected with Leishmania infantum through different routes of inoculation.</a> Parasit Vectors. 9 (1), 181-194 (2016).</li>
<li>Carnielli, J. B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Leishmania+infantum+genetic+marker+associated+with+miltefosine+treatment+failure+for+visceral+leishmaniasis%5BTitle%5D)+AND+%22EBioMedicine%22%5BJournal%5D)">A Leishmania infantum genetic marker associated with miltefosine treatment failure for visceral leishmaniasis.</a> EBioMedicine. 36, 83-91 (2018).</li>
<li>Carnielli, J. B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+resistance+of+Leishmania+infantum+to+miltefosine+contributes+to+the+low+efficacy+in+the+treatment+of+visceral+leishmaniasis+in+Brazil%5BTitle%5D)+AND+%22Am+J+Trop+Med+Hyg%22%5BJournal%5D)">Natural resistance of Leishmania infantum to miltefosine contributes to the low efficacy in the treatment of visceral leishmaniasis in Brazil.</a> Am J Trop Med Hyg. 101 (4), 789-794 (2019).</li>
<li>Moreira, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+continuous+axenic+cultivation+in+Leishmania+infantum+virulence%5BTitle%5D)+AND+%22PLoS+Negl+Trop+Dis%22%5BJournal%5D)">Impact of continuous axenic cultivation in Leishmania infantum virulence.</a> PLoS Negl Trop Dis. 6 (1), e1469(2012).</li>
<li>Fortin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+and+tolerability+of+oleylphosphocholine+%28OlPC%29+in+a+laboratory+model+of+visceral+leishmaniasis%5BTitle%5D)+AND+%22J+Antimicrob+Chemother%22%5BJournal%5D)">Efficacy and tolerability of oleylphosphocholine (OlPC) in a laboratory model of visceral leishmaniasis.</a> J Antimicrob Chemother. 67 (11), 2707-2712 (2012).</li>
<li>Rodrigues, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+gingival+vein+is+a+minimally+traumatic+site+for+multiple+blood+sampling+in+guinea+pigs+and+hamsters%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">The gingival vein is a minimally traumatic site for multiple blood sampling in guinea pigs and hamsters.</a> PLoS ONE. 12 (5), e0177967(2017).</li>
<li>Oliveira, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technical+report%3A+Gingival+vein+punction%3A+A+new+simple+technique+for+drug+administration+or+blood+sampling+in+rats+and+mice%5BTitle%5D)+AND+%22Scand+J+Lab+Anim+Sci%22%5BJournal%5D)">Technical report: Gingival vein punction: A new simple technique for drug administration or blood sampling in rats and mice.</a> Scand J Lab Anim Sci. 36 (2), 109-113 (2009).</li>
<li>Saeij, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioluminescence+imaging+of+Toxoplasma+gondii+infection+in+living+mice+reveals+dramatic+differences+between+strains%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains.</a> Infect Immun. 73 (2), 695-702 (2005).</li>
<li>Thalhofer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+transgenic+Leishmania+parasites+in+a+live+host%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. 41, e1980(2010).</li>
<li>Andriani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+in+vivo+of+anti-Trypanosoma+cruzi+compounds+selected+from+a+high+throughput+screening%5BTitle%5D)+AND+%22PLoS+Negl+Trop+Dis%22%5BJournal%5D)">Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening.</a> PLoS Negl Trop Dis. 5 (8), e1298(2011).</li>
<li>Henriques, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+mice+infected+with+bioluminescent+Trypanosoma+cruzi+unveils+novel+sites+of+infection%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">In vivo imaging of mice infected with bioluminescent Trypanosoma cruzi unveils novel sites of infection.</a> Parasit Vectors. 7, 89(2014).</li>
<li>Henriques, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioluminescent+imaging+of+Trypanosoma+cruzi+infection+in+Rhodnius+prolixus%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">Bioluminescent imaging of Trypanosoma cruzi infection in Rhodnius prolixus.</a> Parasit Vectors. 26 (5), 214(2012).</li>
<li>Sereno, D., Lemesre, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axenically+cultured+amastigote+forms+as+an+in+vitro+model+for+investigation+of+antileishmanial+agents%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents.</a> Antimicrob Agents Chemother. 41 (5), 972-976 (1997).</li>
<li>Lachaud, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Value+of+two+PCR+methods+for+the+diagnosis+of+canine+visceral+leishmaniasis+and+the+detection+of+asymptomatic+carriers%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">Value of two PCR methods for the diagnosis of canine visceral leishmaniasis and the detection of asymptomatic carriers.</a> Parasitology. , 197-207 (2002).</li>
<li>Blood sampling Microsite. , National Centre for the Replacement, Refinement and Reduction of animals in research (NC3RS). 
 <a target="_blank" href=" https://www.nc3rs.org.uk/3rs-resources/blood-sampling/blood-sampling-hamster"> https://www.nc3rs.org.uk/3rs-resources/blood-sampling/blood-sampling-hamster</a> (2021).</li>
<li>Bolhassani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+Leishmania+species%3A+development+of+stable+GFP+expression+and+its+application+for+in+vitro+and+in+vivo+studies%5BTitle%5D)+AND+%22Exp+Parasitol%22%5BJournal%5D)">Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies.</a> Exp Parasitol. 127 (3), 637-645 (2011).</li>
<li>Mendes Costa, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+infection+with+bioluminescent+Leishmania+infantum+axenic+amastigotes+applied+to+drug+discovery%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Murine infection with bioluminescent Leishmania infantum axenic amastigotes applied to drug discovery.</a> Sci Rep. 9 (1), 18989(2019).</li>
</ol>

The authors have no conflicts of interest to disclose.

Blood collection or intravenous injection of substances into hamsters is necessary for various scientific studies. Several methods have been developed to access different collection or inoculation routes directly related to the research objectives19. Due to the hamster anatomy-a short tail and limbs-the intracardiac route is usually the choice for intravenous injection of Leishmania. Depending on the strain used, the intracardiac route proved advantageous as the reference strain L. infantum MHOM/BR/1974/PP75, whose infection occurs over the long term, 6-9 months5. However, it is an inoculum that can lead to bleeding and the death of the animal. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival plexus, mandibular labial vein, that causes less harm to the animal. The animals were infected with the genetically modified reference strain L. infantum MHOM/BR/1974/PP75, which stably expressed the firefly luciferase even after several passages in culture and mice (Figure 1), as related for other Leishmania species transfected by the same integrative plasmid20.
The mandibularis labialis vein or gingival vein is a better route for blood sampling, and for multiple blood collection10,11. However, this is the first demonstration that the gingival vein is a feasible site for intravenous infection by Leishmania in hamsters. In contrast to blood sampling that usually uses a high gauge 26 G needle to avoid blood hemolysis10, this needle gauge was not appropriate for Leishmania inoculation, due to vein perforation, bleeding, and leakage of the inoculum. For Leishmania infection via the maxillary mandibularis vein, a smaller gauge 30G-needle was essential. Another aspect that differentiates the vein puncture to infection via gingival vein is the rate of administration, at approximately 1 &#956;L/s; and to ensure that it is injected into the vessel-the mandibular labial vein, and it is not lodged in the mucosa, subcutaneous or intradermal. Owing to the low blood turnover of the gingival plexus, the 50 &#956;L of a high-density inoculum of axenic amastigotes or promastigotes of L. infantum-Luc, 2 x 109 parasites/mL, had to be inoculated slowly (~ 1 min), and the needle has to be removed by keeping the swab pressed for 1 min to allow the inoculum dispersion in the bloodstream (Figure 2).
For longitudinal evaluation of infection hamsters were infected intraperitoneally (IP) or via the intragingival (IG) route with L. infantum-Luc and were followed up for 50 dpi by the bioluminescence imaging system until euthanasia. Considering that the reference strain PP75 could be less virulent per se and that the super-expression of luciferase could also impact the infection effectiveness and maintain the infection for the long term, a high inoculum of 108 parasites was used for infection. After gingival inoculation of both amastigotes and promastigotes of L. infantum-Luc and evaluation by the bioluminescence imaging system, bioluminescence was restricted to the maxillary region of hamsters 24 h after infection. Indeed, hamsters infected intraperitoneally with amastigotes and promastigotes of L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen (Figure 3, 1 dpi). The continuous decrease in bioluminescent emission along the time, from the first day of infection through the 8th day and until the 22nd dpi in hamsters infected with L. infantum-Luc, was independent of the inoculation route (Table 1 and Figure 4).
However, when the parasite burden in the animal tissues is low, it can be below the detection limit of the bioluminescence imaging system but can be detected and quantified by PCR or qPCR. As already reported, the infection caused by strain PP75 is indeed lower than that of other strains5, and only a few developed clinical signs of the disease due to the genetic variability of the animals. In this study, despite the small number of animals and the low virulence of this strain, the axenic amastigotes demonstrated an advantage at 50 dpi, showing better infection than promastigotes, as demonstrated by PCR (Figure 5). Eight months after infection with amastigotes via the gingival route, parasites could be detected by PCR in the liver (Figure 5) and they also displayed moderate piloerection, orbital tightness, and arched posture.
Axenic amastigotes can be a better choice for infection and follow-up than promastigotes21 and have the advantage of being easy to produce on a large scale. The gingival inoculum is feasible and a better route for intravenous inoculation of compounds and for infection of Leishmania and other pathogens, without damage or swelling at the application site in the mandibula or to the animal health.

The leishmaniasis are considered neglected and re-emerging diseases caused by more than 20 species of Leishmania, endemic in several countries in the four central eco-epidemiological regions: Latin America, North and East Africa, and West and Southeast Asia1. They can be grouped as tegumentary (TL) and visceral leishmaniasis (VL), which is lethal if not treated. The etiological agent of VL in Brazil is Leishmania infantum, and treatment is carried out using pentavalent antimonials or Amphotericin B. These drugs are administered intravenously, have high toxicity, display adverse reactions, and there are cases of resistance2.
Thus, it is necessary to invest in the search for new chemotherapy. Preclinical tests are, in fact, a bottleneck in the discovery of new drugs for VL treatment, depending on the animal model, the strain used, the inoculum route, and other logistical, technical, and operational factors. The golden hamster stands out for its high susceptibility to species of the Viannia and Leishmania subgenera, displaying many of the clinical and immunopathological processes observed in the human disease as observed in previous studies with Leishmania braziliensis3,4. The hamster infected with L. infantum also develops most of the immunopathological processes characteristic of VL in humans and dogs5, such as anemia, leukopenia, thrombocytopenia, and hepatosplenomegaly. In addition, the golden hamster is an outbred animal and does not show a uniform response, reproducing the heterogeneity of clinical manifestations as seen in humans3.
Another aspect to consider for infection outcome is the L. infantum strain and the route of inoculation. Several strains of L. infantum differ in genetic background and susceptibility to treatment2,6,7. Some strains have low parasitic load in the liver and spleen after promastigotes infection8, and axenic amastigotes can be an alternative to improve the infection that is not much explored. Indeed, the intravenous route favors infection and increases the frequency of animals with clinical signs; but intraperitoneal inoculation is the most used. The intracardiac route is the choice for intravenous infection with L. infantum5,8,9. However, in hamsters, intragingival inoculation is an alternative route for intravenous injection, not described as a site for infection. Despite being reported, gingival venipuncture is minimally invasive, allows easy venous access, and causes few local and systemic injuries10. The gingival vein puncture agrees the most with recommendations to maximize the quality and applicability of results while preserving animal well-being11.
Preclinical evaluation of compounds for VL using traditional methods requires more animals, which must be euthanized for histopathological analysis and assessment of parasite load in the tissues. In contrast, the bioluminescent imaging system can speed up preclinical studies and reduce the number of animals. The bioluminescent sites in the infected tissues can be followed up in real time in the same animal for several weeks. Several studies on the standardization of this crucial technological tool have shown its application in studies with mice infected with Trypanosoma cruzi, Leishmania spp., and Toxoplasma gondii12,13,14,15. However, depending on the the parasite burden in tissue, bioluminescence can be underdetected by the in vivo imaging system, which requires evaluation by quantitative PCR of the affected organs. Therefore, we propose to develop a methodology based on the intravenous injection of L. infantum expressing luciferase in the gingival vein of golden hamsters for follow-up by the bioluminescent imaging system and PCR.

<table><tbody><tr><td>1 kb ladder</td><td>Promega</td><td>G5711</td><td></td></tr><tr><td>1-Butanol</td><td>Sigma-Aldrich</td><td>B7906</td><td></td></tr><tr><td>Acetyl coenzyme A</td><td>Sigma-Aldrich</td><td>A2181</td><td></td></tr><tr><td>Agarose, LE, Analytical Grade</td><td>Promega</td><td>V3125</td><td></td></tr><tr><td>aprt reverse primer A1715&lt;br/&gt; aprt reverse primer A1715</td><td>Jena Bioscience</td><td>PM-111</td><td></td></tr><tr><td>BamHI 10 U/mL</td><td>Promega</td><td>R6021</td><td></td></tr><tr><td>BglII 10 U/mL</td><td>Promega</td><td>R6071</td><td></td></tr><tr><td>brain and heart infusion (BHI)</td><td>Sigma-Aldrich</td><td>53286</td><td></td></tr><tr><td>Cetamin (Ketamine hydrochloride 10%)</td><td>Syntec</td><td>-</td><td>Veterinary use. Anesthetic. Injectable solution containing a 10 mL vial of 10% ketamine hydrochloride.</td></tr><tr><td>Dextrose Glucose, BD Diagnostics</td><td>Difco</td><td>215530</td><td></td></tr><tr><td>D-luciferin potassium salt</td><td>Promega</td><td>E1601</td><td></td></tr><tr><td>DMEM low glucose</td><td>Sigma-Aldrich</td><td>D6046</td><td></td></tr><tr><td>eletroporador Gene Pulser Xcell&amp;nbsp;</td><td>BioRad Laboratories</td><td></td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>vitrocell/embriolife</td><td></td><td></td></tr><tr><td>Ficoll-plaqueTM PLUS&amp;nbsp;</td><td>Cytiva</td><td>17144003</td><td></td></tr><tr><td>Gene Pulser/MicroPulser Electroporation Cuvettes, 0.2 cm gap&lt;br/&gt; 1652082</td><td>Bio-Rad</td><td>1652082</td><td></td></tr><tr><td>Gene Pulser/MicroPulser Electroporation Cuvettes, 0.4 cm gap&lt;br/&gt; 1652081&amp;nbsp;</td><td>Bio-Rad</td><td>1652081</td><td></td></tr><tr><td>GoTaq Platinum polymerase</td><td>Fischer Scientific</td><td>10-966-034&amp;nbsp;</td><td></td></tr><tr><td>GoTaq DNA Polymerase</td><td>Promega</td><td>M3001</td><td></td></tr><tr><td>Hemin powder</td><td>inlab</td><td></td><td></td></tr><tr><td>HEPES buffer</td><td>Sigma-Aldrich</td><td>H3375&lt;br/&gt; Sigma-Aldrich</td><td></td></tr><tr><td>hyg forward primer A3804</td><td>Jena Bioscience</td><td>PM-109</td><td></td></tr><tr><td>Hygromycin</td><td>Sigma-Aldrich</td><td>H3274</td><td></td></tr><tr><td>ISOFORINE</td><td>Cristalia</td><td></td><td>Inhalation solution in packs containing 1 bottle of 100 and 240 mL of isoflurane</td></tr><tr><td>IVIS Lumina</td><td>Perkin Elmer</td><td>ISO838N4625</td><td></td></tr><tr><td>JM109 Competent Cells</td><td>Promega</td><td>&amp;nbsp;L2005</td><td></td></tr><tr><td>L- Glutamin</td><td>Sigma-Aldrich</td><td>G8540&lt;br/&gt; Sigma-Aldrich</td><td></td></tr><tr><td>Lambda DNA/HindIII Marker</td><td>Thermo Fischer Scientific</td><td>SM0101</td><td></td></tr><tr><td>L-cystein</td><td>Sigma-Aldrich</td><td>168149&lt;br/&gt; Sigma-Aldrich</td><td></td></tr><tr><td>Living Image software</td><td>Perkin Elmer</td><td>-</td><td></td></tr><tr><td>NotI 10 U/mL</td><td>Promega</td><td>R6431</td><td></td></tr><tr><td>Phenol/Chloroform/Isoamyl Alcohol, 25:24:1 (v/v), Molecular Biology Grade</td><td>Sigma-Aldrich</td><td>516726</td><td></td></tr><tr><td>Phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>plasmid pLEXSY-hyg2&amp;nbsp;</td><td>Jena Bioscience</td><td>EGE-232</td><td></td></tr><tr><td>Proteinase K</td><td>Promega</td><td>V3021&amp;nbsp;</td><td></td></tr><tr><td>QIAGEN Plasmid Midi Kit&amp;nbsp;</td><td>Qiagen</td><td>12143</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>QIAquick PCR &amp;amp; Gel Cleanup Kit</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>Schneider&#039;s medium&amp;nbsp;</td><td>Gibco</td><td>21720-024</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S9763&lt;br/&gt; Sigma-Al</td><td></td></tr><tr><td>SpectraMax2 microplate reader</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>ssu forward primer F3001&lt;br/&gt; primer F3001</td><td>Jena Bioscience</td><td>PM-105</td><td></td></tr><tr><td>ssu reverse primer F3002&lt;br/&gt; ssu reverse primer F3002</td><td>Jena Bioscience</td><td>PM-104</td><td></td></tr><tr><td>Steady-Glo Luciferase Assay System</td><td>Promega</td><td>E2510</td><td></td></tr><tr><td>SwaI 10 U/mL</td><td>Thermo Scientific</td><td>ER1241</td><td></td></tr><tr><td>T4 DNA ligase 1 U/mL</td><td>Promega</td><td>M1801</td><td></td></tr><tr><td>T4 fast ligation system</td><td>Promega</td><td>M8221</td><td></td></tr><tr><td>Thermal cycler</td><td>Applied Biosystems</td><td>Veritiy 96 well plate</td><td></td></tr><tr><td>TRITON X-100</td><td>Sigma-Aldrich</td><td>T8787&lt;br/&gt; Sigma-Aldrich</td><td></td></tr><tr><td>Tryptic Soy Broth (Soybean-Casein Digest Medium)</td><td>Difco-BD</td><td>211823</td><td></td></tr><tr><td>Ventilated racks&amp;nbsp;</td><td>Alesco</td><td></td><td></td></tr><tr><td>With Earle&amp;prime;s salts and L-glutamine, without sodium bicarbonate, powder,&amp;nbsp;</td><td>Merck</td><td>M5017</td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Xilazina (Xylazine hydrochloride 2%)</td><td>Syntec</td><td>-</td><td>Veterinary use. Sedative, analgesic and myorelaxant. Injectable solution containing a 10 mL vial of 2% xylazine hydrochloride.</td></tr><tr><td>Zero Blunt TOPO PCR Cloning Kit&amp;nbsp;</td><td>Thermo Fischer&amp;nbsp;</td><td>451245</td><td></td></tr></tbody></table>

in vivo imaging of leishmania infantum-infected hamsters by gingival inoculation of axenic amastigotes expressing luciferase

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Stable expression of luciferase in L. infantum 
Genetically modified L. infantum was produced using the plasmid of the pLEXSY line, which integrates into the genome of Leishmania in the 18S rRNA (ssu) ribosomal locus, whose transcription is driven by RNA polymerase I. Thus, L. infantum-Luc clones were evaluated for plasmid integration in the genome of Leishmania, and for stable expression by bioluminescence emission in vitro. The highly expressing clone displaying bioluminescence &gt;120 fold above the background was chosen for evaluation of genomic integration by PCR. See Figure 1 for agarose gel electrophoresis of PCR products to evaluate plasmid integration in the genome and bioluminescence emission (RLU) of promastigotes of the L. infantum-Luc clone. Fragments of the expected size were obtained from each PCR; one product of approximately 1,1 kb (5'ssu - utr1), and another of 1.8 kb (hyg- 3'ssu) were amplified from the genome of L. infantum- Luc (Figure 1A), confirming the integration of the plasmid cassette and the luciferase gene in the ssu locus of L. infantum genome.
The firefly luciferase expression was also evaluated in promastigotes of L. infantum-Luc by bioluminescence emission (RLU) in the microplate reader, as described in protocols, section 2. Even after several passages in culture and in BALB/c mice for 5 days, it maintained the level of bioluminescence; 569.3 ± 19.5 for the wild type background, and 59361.9 ± 2673.3 (n = 2) for cloned L. infantum- Luc (Figure 1B). Thus, the L. infantum-Luc clone stably expressing firefly luciferase, was used to infect hamsters via intragingival or intraperitoneal routes.
Intravenous inoculum in the gingival vein 
To inoculate the Leishmania into the bloodstream of hamsters, care should be taken to minimize vein perforation, bleeding and leakage of the inoculum. Thus, the lower lip has to be pulled down gently to expose the gingival vein (Figure 2A); and a smaller gauge 30 G needle has to be used to avoid excessive perforation of the vein. The needle has to be positioned with the bezel facing up to insert into the vein in a proper angle (Figure 2B). Indeed, to ensure that the needle was injected into the vessel-the mandibular labial vein, the syringe plunger has to be pulled down until blood is aspirated into the needle barrel (Figure 2C). Before needle removal, light pressure has to be applied with a cotton swab to promote hemostasis (Figure 2D).
Longitudinal evaluation by bioluminescence imaging 
Hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc were followed up until the 50th dpi and were evaluated by bioluminescence imaging until 22 dpi (Figure 3). Images were acquired 2 h after intraperitoneal infection with 108 parasites in the peritoneal cavity; images were acquired for 30 s or 1 min exposition binning medium. The bioluminescence signal was &gt;65 fold more intense in the abdomen in amastigote-infected animals (4.6 × 105 ± 3.7 × 105) than promastigote-infected ones (6.8 × 103 ± 3.8 × 103) (Table 1), which demonstrates that amastigotes differentiated in vitro are more bioluminescent than the metacyclic promastigotes at the stationary phase and purified in the Ficoll cushion.
One day post infection (1 dpi), bioluminescence images were acquired for 3 min of exposition (Figure 3). There was a 45% decrease in the bioluminescence signal in the abdominal region in promastigote-infected hamsters and 70% decay in amastigote-infected ones (Figure 3), which suggests that amastigotes had degraded to a greater extent than metacyclic promastigotes (Table 1 and Figure 4). One week after infection (8 dpi), promastigote-infected animals sustained the bioluminescence signal (3.3 × 103 ± 5 × 103). However, the bioluminescence emission in amastigote-infected hamsters dropped off 95%, from 1.3 × 105 ± 1.1 × 105 to 6.7 × 103 ± 7.5 × 103 (Table 1) and reached the same level of promastigote-infected hamsters. Three weeks after infection (22 dpi), a bioluminescence signal was acquired for 5 min of exposition and binning large (Figure 3); the signal was much lower for promastigotes and amastigotes-infected animals (Table 1 and Figure 4).
Another group of hamsters was infected via the intragingival route with amastigotes and promastigotes of L. infantum-Luc (108); bioluminescence emission was observed in the maxillary region (Figure 3). The follow-up started 1 day after infection, and amastigote-infected hamsters displayed more bioluminescence signal and radiance (7.3 × 103 ± 4.1 × 103) than promastigote-infected ones (1 × 103 ± 5.7 × 102). One week after infection (8 dpi), a 36% drop in bioluminescence signal was observed in promastigotes-infected animals and 90% in the amastigote-infected hamsters; radiance varied from 7.3 × 103 ± 4.1 × 103 to 7.8 × 102 ± 5.6 × 102 (Table 1). Three weeks after infection (22 dpi), a bioluminescence signal was also acquired for 5 min of exposition and binning large (Figure 3). The bioluminescence signal was similar and low for promastigote- and amastigote-infected animals (Table 1 and Figure 4) in the head of animals infected at the gingiva and was not observed dispersion of the infection to the abdominal region by bioluminescence signal.
Evaluation of infection in tissues and organs by PCR 
We performed conventional PCR to investigate infection in specific organs, such as liver, spleen and lymph nodes, that could be below the detection limit of the in vivo imaging. The target region of kDNA was more specific for L. infantum DNA amplification in infected tissues and organs, and the PCR for the enzyme GAPDH from the hamster was a control of DNA integrity and PCR reaction. Only the samples that were amplified for GAPDH were considered in the analysis. Thus, by PCR, two of three hamsters infected via the intraperitoneal route with axenic amastigotes displayed infection in tissues and organs; animal two (A2) in the spleen and animal three (A3) in the liver, at 50 dpi (Figure 5A). One hamster was infected intraperitoneally with axenic promastigotes; animal three (A3) (Figure 5B) displayed amplification in the lymph node. Hamsters inoculated intragingivally with promastigotes or axenic amastigotes at 50 dpi could not display a clear amplification-just a band in the liver of animal one (A1) infected with promastigotes (Figure 5C). Notably, we had three animals maintained for 8 months whose inoculum delivered by the intragingival route with amastigotes leaked slightly during the injection. Two of three animals displayed a clear infection in the liver, animals one and two (Figure 5D).
<img alt="Gel electrophoresis and bioluminescence chart; DNA bands and L. infantum luciferase expression." class="xfigimg" src="/files/ftp_upload/67050/67050fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Evaluation of the Leishmania infantum-Luc clone by PCR and bioluminescence emission. (A) Agarose gel electrophoresis of PCR products to evaluate plasmid integration in the genome: lane 1 - 1 kb DNA ladder; PCR of L. infantum-Luc genomic DNA, lane 2 - 5'ssu - utr1 (1.1 kb) and lane 3- hyg- 3'ssu (1.8 kb); PCR of L. infantum-wt genomic DNA, lanes 4 and 5.(B) Bioluminescence emission (RLU) of promastigotes of L. infantum-Luc clone (106) in the microplate reader. <a href="https://www.jove.com/files/ftp_upload/67050/67050fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rodent dental procedure showing four stages; syringe application for teeth cleaning and examination." class="xfigimg" src="/files/ftp_upload/67050/67050fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Intravenous injection of Leishmania infantum-Luc into the gingival vein. (A) The hamster was placed in dorsal decubitus, and the lower lip was pulled down. (B) A thinner needle (8 x 0.30 mm) coupled to a 1 mL syringe was positioned below the lower incisors along the middle line between the pair of teeth at an angle of 25º and inserted 2-4 mm into the mandibularis labialis vein. (C) Inoculation of 50 µL (108) of amastigotes or promastigotes in PBS. (D) Hemostasis using a cotton swab and applying light pressure to the inoculation site. <a href="https://www.jove.com/files/ftp_upload/67050/67050fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Leishmania infection progression in mice, fluorescence imaging, intraperitoneal, intragingival methods." class="xfigimg" src="/files/ftp_upload/67050/67050fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Follow-up by in vivo bioluminescence imaging. Representative images of one animal per group: Infected via intraperitoneal (upper panels) or intragingival (lower panels), with amastigotes or promastigotes of L. infantum-Luc, for 1, 8, and 22 dpi. Red ROI representing the probed regions at the abdomen and head, for intraperitoneal or intragingival infection, respectively. Data show that at 1 dpi, all animals displayed bioluminescence signal in the abdomen or mandible. The signal was dropping after 8 dpi and was almost undetectable in any group at 22 dpi. <a href="https://www.jove.com/files/ftp_upload/67050/67050fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Promastigote and amastigote infection radiance graph; data analysis of days post-infection (dpi)." class="xfigimg" src="/files/ftp_upload/67050/67050fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Comparative analysis of radiance from bioluminescence images. Radiance quantification photons.sec-1.cm-2.sr-1 was performed in the abdomen or head of hamsters with manual ROI measurement tools. Average background ROI was subtracted from measurement ROI to remove any spurious signal. <a href="https://www.jove.com/files/ftp_upload/67050/67050fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electrophoresis results, DNA bands at 564 bp and 125 bp, gel analysis for gene expression comparison." class="xfigimg" src="/files/ftp_upload/67050/67050fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: PCR amplification of kDNA. (A) IP-AMA, hamsters infected via intraperitoneal route with amastigote, 50 dpi (n = 3); (B) IP-PRO, infected via intraperitoneal route with promastigotes, 50 dpi (n = 3); (C) IG-AMA, infected via intragingival route with amastigote (n = 2), IG-PRO, infected via intragingival route with promastigote (n = 2); (D) IG-AMA, infected via intragingival route with amastigote, 8 months post infection (n = 3). NI, uninfected hamsters as negative control (n = 2); C- genomic DNA of L. infantum-Luc, positive control of PCR. Tissues and organs: 1- spleen, 2- liver, 3- lymph nodes. mw- molecular weight marker, arrows indicate the lower molecular weight bands. A1- animal one, A2- animal two, and A3- animal three. <a href="https://www.jove.com/files/ftp_upload/67050/67050fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td style="min-width: 60px;">dpi</td>
			<td colspan="2" style="min-width: 60px;">Amastigotes IP</td>
			<td style="min-width: 60px;"></td>
			<td colspan="2" style="min-width: 60px;">Promastigotes IP</td>
			<td style="min-width: 60px;"></td>
			<td colspan="2" style="min-width: 60px;">Amastigotes IG</td>
			<td style="min-width: 60px;"></td>
			<td colspan="2" style="min-width: 60px;">Promastigotes IG</td>
			<td style="min-width: 60px;"></td>
		</tr>
		<tr>
			<td style="min-width: 60px;"></td>
			<td style="min-width: 60px;">Mean</td>
			<td style="min-width: 60px;">SD</td>
			<td style="min-width: 60px;">N</td>
			<td style="min-width: 60px;">Mean</td>
			<td style="min-width: 60px;">SD</td>
			<td style="min-width: 60px;">N</td>
			<td style="min-width: 60px;">Mean</td>
			<td style="min-width: 60px;">SD</td>
			<td style="min-width: 60px;">N</td>
			<td style="min-width: 60px;">Mean</td>
			<td style="min-width: 60px;">SD</td>
			<td style="min-width: 60px;">N</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">0</td>
			<td style="min-width: 60px;">4.6 x 105</td>
			<td style="min-width: 60px;">3.7 x 105</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">6.8 x 103</td>
			<td style="min-width: 60px;">3.8 x 103</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">-</td>
			<td style="min-width: 60px;">-</td>
			<td style="min-width: 60px;">-</td>
			<td style="min-width: 60px;">-</td>
			<td style="min-width: 60px;">-</td>
			<td style="min-width: 60px;">-</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">1</td>
			<td style="min-width: 60px;">1.3 x 105</td>
			<td style="min-width: 60px;">1.1 x 105</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">3.8 x 103</td>
			<td style="min-width: 60px;">5.5 x 103</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">7.3 x 103</td>
			<td style="min-width: 60px;">4.1 x 103</td>
			<td style="min-width: 60px;">2</td>
			<td style="min-width: 60px;">1.0 x 103</td>
			<td style="min-width: 60px;">5.7 x 102</td>
			<td style="min-width: 60px;">2</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">8</td>
			<td style="min-width: 60px;">6.7 x 103</td>
			<td style="min-width: 60px;">7.5 x 103</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">3.3 x 103</td>
			<td style="min-width: 60px;">5.0 x 103</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">7.8 x 102</td>
			<td style="min-width: 60px;">5.6 x 102</td>
			<td style="min-width: 60px;">2</td>
			<td style="min-width: 60px;">6.4 x 102</td>
			<td style="min-width: 60px;">8.2 x 101</td>
			<td style="min-width: 60px;">2</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">22</td>
			<td style="min-width: 60px;">7.3 x 101</td>
			<td style="min-width: 60px;">8.5 x 101</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">9.9 x 101</td>
			<td style="min-width: 60px;">8.6 x 101</td>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">4.6 x 102</td>
			<td style="min-width: 60px;">7.5 x 101</td>
			<td style="min-width: 60px;">2</td>
			<td style="min-width: 60px;">5.0 x 102</td>
			<td style="min-width: 60px;">1.5 x 102</td>
			<td style="min-width: 60px;">2</td>
		</tr>
	</tbody>
</table>
Table 1: Raw data from comparative radiance analysis of bioluminescence images. Radiance quantification average photons.sec-1.cm-2.sr-1 by group and route. Abbreviations: dpi = days post infection; SD = standard deviation; N = sample size.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

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Research

JoVE Journal

Biology

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Using Luciferase to Image Bacterial Infections in Mice

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of  Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes,  transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the...

An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

The protocol provided here describes the detailed steps to perform drug efficacy studies in a bioluminescent model of Trypanosoma cruzi infection, with a focus on data acquisition, analysis, and interpretation. Troubleshooting and quality control procedures to minimize technical issues are also provided.

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

Learn bioluminescent imaging of Leishmania infantum infection in golden hamsters using gingival vein inoculation of axenic amastigotes. This method enables real-time tracking of parasite burden for preclinical research.

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

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