Bioluminesens Imaging å oppdage sent stadium smitte av sovesyke
Summary
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.
Abstract
Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.
Introduction
Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the vector-borne protozoan parasites of the Trypanosoma brucei spp1. Estimated numbers of current cases is fewer than 7 thousand every year with almost 70 million people exposed to the risk of the parasite infection within the African continent. The disease, which is most often lethal if left untreated, comprises an early hemolymphatic stage where parasites are present in the blood, progressing to the late stage when parasites invade the central nervous system (CNS) and are no longer susceptible to treatment by early stage trypanosomal drugs2. The current drug therapies for late-stage HAT have both complex, prolonged, treatment regimens and severe adverse effects as well as reported resistance, therefore research into new drug therapies is imperative3,4.
The study of late-stage human African trypanosomiasis (HAT) within traditional mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden5. The animal infective strain T. b. brucei is used as the study model of trypanosomiasis with the late stage appearing 21 days post infection (dpi). To monitor the wild type nonbioluminescent parasite infection in the mouse model, peripheral blood films or quantitative PCR are the only methods to determine parasite burden. For parasite burden in the brain, the mouse needs to be culled, brain excised and qPCR carried out on tissues, making it impossible to track parasites through multiple time points in the late stage infection. This results in the inability to follow real-time infections within the central nervous system (CNS).
In vivo bioluminescence imaging (BLI) can provide highly sensitive, non-invasive detection of parasite dissemination and disease progression in a mouse model that can be followed in a single animal for the entirety of the experiment6. BLI is based on the emission of light in the visible spectrum produced by a luciferase-catalyzed reaction. The emitted photons are then detected by a charge coupled device (CCD) camera7. For this purpose, the pathogen is genetically modified to express a luciferase protein and the substrate, luciferin, is introduced at time points of interest by injection. The main advantage of this method is the ability to carry out longitudinal studies, in which the same animal can be imaged several times with minimal adverse effects. The acquired bioluminescence signal can be quantified, thus indicating the pathogen burden.
The optimization and validation of a red-shifted bioluminescent T. b. brucei has enabled the investigation of the late stage infection through non-invasive procedures, detecting parasites earlier than blood film microscopy and greatly reducing the time, cost and numbers of animals needed to study CNS infection and drug screening in late-stage trypanosomiasis8,9. In this protocol we demonstrate infection of mice with bioluminescent trypanosomes and how to then visualize the parasites in vivo for quantification of disease progression and CNS penetration.
Protocol
Representative Results
Discussion
Utviklingen av en bioluminescent T. b. brucei GVR35 belastning tillater visualisering av en trypanosom infeksjon fra tidlig til sent stadium. Tidligere smittemodeller ikke var i stand til å oppdage sent stadium, når parasitter er i hjernen, i sanntid fra blod film mikroskopi, og krevde culling og fjerning av hjerner fra den infiserte mus for å bestemme parasitt byrde 12. Bioluminescence reduserer inter-mus variasjon som et enkelt muse kan spores gjennom helheten av infeksjonen og en rask kvalitati…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Vi takker John Kelly og Martin Taylor (London School of Hygiene & Tropical Medicine) for å gi T. b. brucei GVR35-VSL-2 og Dr. Andrea Zelmer (LSHTM) for råd om in vivo imaging. Dette arbeidet ble støttet av Bill og Melinda Gates Foundation Global Health Program (tilskudd nummer OPPGH5337).
Materials
| PBS | Sigma, UK | P4417 | tablets pH 7.4 |
| Glucose | Sigma, UK | G8270 | 99.5% (molecular) grade |
| Ammonium chloride | Sigma, UK | A9434 | 99.5% (molecular) grade |
| Heparin (lithium salt) | Sigma, UK | H0878 | |
| Hi-FCS | Gibco, Life Technologies, UK | 10500-064 | 500 ml |
| DPBS | Sigma, UK | D4031 | Sterile filtered |
| Mr. Frosty | Nalgene, UK | ||
| Giemsa | Sigma, UK | G5637 | |
| D-Luciferin | Perkin Elmer, UK | ||
| Sigma, UK | 115144-35-9 | ||
| Diminazene aceturate | Sigma, UK | D7770 | Analytical grade |
| IVIS Lumina II | Perkin Elmer, UK | other bioimagers available e.g. from Bruker, Kodak | |
| Living Image v. 4.2 | Perkin Elmer, UK | proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own | |
| 1 ml syringe | Fisher Scientific, UK | 10142104 | |
| 20 ml syringe | Fisher Scientific, UK | 10743785 | |
| 25G Needles | Greiner Bio-one | N2516 | |
| 21G Needles | Greiner Bio-one | N2138 | |
| Twin-frosted microscope slide | VWR, UK | 631-0117 | |
| 1.5 ml microcentrifuge tube | StarLab, UK | I1415-1000 | |
| 7 ml Bijou tube | StarLab, UK | E1412-0710 | |
| Mouse restrainer | Sigma, UK | Z756903 | our restrainer was made in-house, this is a similar model |
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