Agradecemos a John Kelly y Martin Taylor (Escuela de Higiene y Medicina Tropical) para proporcionar T. segundo. brucei GVR35-VSL-2 y el Dr. Andrea Zelmer (LSHTM) para el asesoramiento en imágenes in vivo. Este trabajo fue apoyado por el Programa Bill y Melinda Gates, la Fundación Mundial de la Salud (número de concesión OPPGH5337).

Ética Todo el trabajo se llevó a cabo bajo la aprobación del Ministerio del Interior británico los Animales (Procedimientos Científicos) de la Ley de 1986 y la London School of Higiene y Medicina Tropical de Bienestar Animal y la Junta de Revisión de Ética. LLEGA directriz se siguen en este informe. 1. En Vivo La aprobación de Trypanosoma brucei brucei bioluminiscente <ol><li> Retirar una acción criopreservados (denominado estabilizado) de T. segundo. brucei cepa GVR35-VSL-2 (disponible en el profesor John Kelly en la London School of Higiene y Medicina Tropical través MTA) de 9 de nitrógeno líquido y se deja que se equilibre a temperatura ambiente. Diluir estabilizado a 2 x 10 4 tripanosomas / ml en pH estéril salina tamponada con fosfato de glucosa (PBS-G) 8,0 (1 L PBS + 15 g de glucosa) 10. Nota: T. segundo. brucei GVR35-VSL-2 no es infecciosa para los humanos y no requiere licencia SAPO instalaciones para llevar a cabo los experimentos. </li><li> inject un 20-25 g ratón hembra CD1 con 0,2 ml diluidos tripanosomas por vía intraperitoneal (ip) con una aguja de 25 G. Este es el &quot;ratón donante&quot;. Coloque el ratón infectado en un libre de patógenos (SPF) -cage y alimentación ad libitum específico. Nota: Los ratones CD1 son una cepa no consanguínea y T. segundo. brucei infecciones GVR35 están bien caracterizados en esta cepa. Sin embargo, este protocolo también es aplicable a cepas puras o modificadas genéticamente, pero es importante tener en cuenta que el color de la piel puede afectar a la sensibilidad de formación de imágenes 6,9. </li><li> Monitorear la parasitemia periférica mediante el muestreo de sangre. Coloque el ratón en un retenedor de plástico y tomar 5 l de sangre de la cola por punción venosa con aguja 21 G o sangría y mezclar 1/10 con cloruro de amonio estéril 0,85% para lisar las células rojas de la sangre y permitir el recuento más fácil. </li><li> Contar la sangre diluida en un hemocitómetro Neubauer. <ol><li> Cargar la sangre diluida en ambas cámaras. Contar todo el centro grIdentificación de una cámara para dar N tripanosomas. La concentración de tripanosomas en la sangre = n x 10 4 tripanosomas / ml, ajuste para cualquier factor de dilución y calcular la media de las dos cámaras. Nota: Después de aproximadamente 5-7 días después de la infección, la parasitemia periférica debe elevarse lo suficiente como para llevar a cabo una infección a una concentración de 2 x 10 4 tripanosomas / ml. </li></ol></li><li> Cuando los niveles de tripanosomas son suficientes para el número de ratones requeridos alta, pasar al paso 1.6. Un nivel de parasitemia de 6 x 10 4 tripanosomas / ml en un ratón donante es suficiente para la infección de 10 ratones. Nota: Para tamaños de los grupos estadísticamente significativas, n = 6 para cada grupo de tratamiento de los ratones se necesitan 8. Para otros modelos de infección, tendría que ser aplicada para determinar los tamaños de grupo apropiados los cálculos de potencia. </li><li> Coloque los ratones en una caja caliente (a 28-30 ° C durante aproximadamente 5-10 minutos) para permitir que las venas de la cola se dilaten.Para inducir la anestesia terminal, administrar 20 mg / kg iv pentobarbital con una aguja 25 G. Confirmar ratón se anestesia presionando suavemente las almohadillas de las patas para asegurar que no hay reflejo de retirada. </li><li> Heparinizar una aguja 21 G por alícuotas de 5 ml de heparina de 5.000 unidades USP / ml en un tubo de Bijou y chupando dos veces en y fuera de la aguja unida a una jeringa de 1 ml. </li><li> Tome el heparinizada 21 G aguja y jeringa de 1 ml, y realizar una punción cardiaca en el ratón paso anestesiado mediante la inserción de la aguja en el centro debajo de la caja torácica (una gota de sangre se acumule en la base de la aguja) y tire suavemente hacia atrás el émbolo a fin de no colapsar la cámara del corazón. Recoger un mínimo de 0,7 ml de sangre. </li><li> Diluir la sangre infectada para producir un inóculo de 2 x 10 4 tripanosomas / ml en PBS-G pH 8,0 como paso 1.1 anteriormente. Nota: En este punto, la sangre restante se pueden criopreservar para producir estabilizado por mezcla con 8% de glicerol, 20% inactivado por calor ca fetalsuero LF (hi-FCS) y la sangre infectada del 72%. Poco a poco se congelan a -80 ° C utilizando un lento-freezing contenedor para lograr una velocidad de enfriamiento de -1 ° C / min, antes de transferir a nitrógeno líquido. </li></ol> 2. La infección de ratones experimentales <ol><li> Coloque el CD1 ratones hembra (~ 21-25 g) en una caja de calentamiento para calentar suavemente para dilatar las venas. Coloque los ratones calentado en un sistema de retención de plástico e inyectar 0,2 ml de inóculo diluido por vía intravenosa con una aguja de 25 G en la vena caudal, equivalente a 4.000 tripanosomas por ratón. Colocar presión en el sitio de la inyección después de detener la hemorragia. Nota: la infección intraperitoneal es una ruta válida de la infección y se ha utilizado en estudios previos 10, pero hemos hallado que este menos reproducible que la vía intravenosa. </li><li> Selección aleatoria de los ratones infectados y la jaula en grupos de 3 en jaulas ventiladas SPF. Como control, se inyectan ratones hembra CD1 de tipo salvaje con parásito no bioluminiscente, T. segundo. brucei GVR35 (n =3). </li><li> Monitorear la infección a través del cine Microscopía de Sangre <ol><li> Spot 5 l de sangre de la punción venosa o sangrías en un portaobjetos de vidrio y realizar un frotis de sangre (utilizando una segunda lámina de vidrio empuje suavemente la gota de sangre a través de la corredera para producir una película delgada). Permitir que las diapositivas se sequen al aire. </li><li> Fijar la muestra de sangre con metanol al 100% y tinción con Giemsa durante 10 minutos antes de enjuagar con H2O destilada y dejar secar al aire. En el objetivo 100X contar el número de tripanosomas en 10 campos de visión (FOV). Nota: la filmación de la sangre se lleva a cabo junto con imágenes no invasivo animal entero tan pronto como un día después de la infección. Cuando se procesa un número de animales, se prefiere este método de cuantificación tripanosoma sobre conteo hemocitómetro que las muestras se pueden almacenar a temperatura ambiente y se contaron después. </li></ol></li></ol> 3. La bioluminiscencia de imágenes para realizar el seguimiento de Infecciones Nota: Para controlar la infección, toda animde formación de imágenes al no invasiva se puede utilizar. <ol><li> Preparar una solución madre de D-luciferina, el sustrato de luciferasa de luciérnaga, en Dulbecco salina tamponada con fosfato (DPBS) a 30 mg / ml. Filtro de esterilizar y congelar en alícuotas a -20 ° C. Nota: D-luciferina es sensible a la luz, por lo tanto, protegerlo de la luz envolviendo alícuotas en papel de aluminio. No congelar y descongelar repetidamente luciferina, ya que puede afectar a la actividad 11. </li><li> Calentar una alícuota de D-luciferina a temperatura ambiente. Retirar cada ratón de la jaula (incluyendo los ratones de control infectados con nonbioluminescent cepa del parásito de tipo salvaje) e inyectar 150 mg / kg de la D-luciferina calentado (diluido en DPBS) por vía intraperitoneal con una aguja 25 G. Coloque los ratones en una cámara de isofluorano durante aproximadamente 5 min para inducir la anestesia con isofluorano al 2% / O 2 mezcla a 2,5 L / min. Nota: Los ratones se vuelven inmóviles cuando están bajo anestesia. Para el generador de imágenes se utiliza aquí, 3 ratones pueden ser procesados ​​a la vez. Si los ratones son anesthetracterizado por más de 30 min, oftálmica ungüento lágrima artificial se puede aplicar a evitar que los ojos de los ratones de la desecación. </li><li> Para el generador de imágenes se describe aquí, abra el software e inicializar la máquina utilizando el software según las instrucciones del fabricante. Adquirir imágenes una vez que la cámara y placa calentada han alcanzado la temperatura. Nota: Este instrumento no está normalmente apagado, permitiendo a la máquina para realizar las calibraciones de fondo durante la noche. En el caso de que se debe reiniciarse, se debe realizar una verificación de la calibración en primer lugar. </li><li> Coloque los ratones anestesiados en el generador de imágenes (véase 3.5). Utilice separadores negros grandes entre los ratones para minimizar sangrar a través de cualquier señal bioluminiscente brillante. los ratones de imagen utilizando un conjunto de tiempos de exposición; 1, 3, 10, 30, 60 y 180 segundos, con binning medio, 1 f / stop y un filtro abierto, y el campo visual E (12,5 x 12,5 cm 2). Nota: Los ratones de control infectados con los parásitos de tipo salvaje GVR35 nonbioluminescent proporcionan un backgrvalor bioluminiscencia ound. Desde un experimento de cinética de luciferina (Figura 6) con este modelo, hemos encontrado que los picos de señal de bioluminiscencia en 10 minutos después de la administración y de formación de imágenes se lleva a aproximadamente 5 min para 3 ratones. Tome esta escala de tiempo en la consideración al anestesiar y imágenes de los ratones. </li><li> Para asegurar la formación de imágenes a fondo de los ratones, girar para obtener un ventral, dorsal y lateral. Mantener la coherencia en el orden de orientación para formación de imágenes entre los animales secuenciales. </li><li> Después de proyección de imagen, permiten ratones se recuperen de la anestesia bajo observación antes de volver a SPF-jaula. </li><li> Continuar la formación de imágenes ratones cada 7 días hasta el día 21 después de la infección. Debe haber un aumento constante de la señal de la bioluminiscencia durante todo el animal. </li><li> En D21, imagen y frotis de sangre de los ratones como se describió anteriormente, y la dosis de los ratones con 40 mg / kg por vía intraperitoneal diminazeno aceturato. Este fármaco se borrará la parasitemia periférica, pero no va a cruzar la barrera sangre-Brain barrera, y por lo tanto permite una mejor visualización de la infección del SNC. Nota: diminazeno aceturato es un fármaco bien establecido utilizado para tratar las primeras etapas de la tripanosomiasis animal. </li><li> Continuar imágenes y controlarse en D28 y D35. </li></ol> 4. Confirmación de la infección del SNC <ol><li> En D35, los ratones de imagen indicados anteriormente (pasos 3.1-3.4). </li><li> Después de proyección de imagen, permiten ratones se recuperen de la anestesia y ratones Exsanguinate como se detalla en el paso 1.6 hasta 1.8, la recogida de sangre para su posterior análisis. </li><li> Después de punción cardíaca, perfundir los ratones con 20 ml de PBS (opcional: añadir 3 mg / ml de luciferina para solución de perfusión) a través del corazón para limpiar la sangre de los órganos y de volver a introducir la etiqueta luciferina que podrían haber desaparecido de la región del cerebro durante el intervalo de tiempo desde el paso 4.1. </li><li> eliminar suavemente el cerebro, mediante el uso de tijeras curvas, el corte de la base alrededor de la circunferencia del cráneo, y el levantamiento de la parte superior del cráneo, para permitir que el cerebropara ser levantado suavemente con fórceps curvos. </li><li> Coloque el cerebro extirpado en una sección de plástico negro y pipeta de 50 l de 15 mg / ml de luciferina sobre el órgano antes de la imagen, para asegurar luciferina adecuada se ha añadido al cerebro extirpado. Imagen utilizando la configuración que el anterior. Esto demostrará la localización de la infección a los hemisferios cerebrales. Nota: cuantificación Aguas abajo de parasitemia en el cerebro extirpado se puede llevar a cabo por qPCR como se describió previamente 8. Otras aplicaciones posteriores alternativas podrían incluir la histología o inmunohistoquímica para identificar los parásitos en el tejido cerebral después de la fijación. </li></ol> 5. La cuantificación de la bioluminiscencia Imaging Nota: La bioluminiscencia se puede cuantificar utilizando la región de interés (ROI) con el software de imágenes y corregida para el fondo bioluminiscencia. <ol><li> Con la herramienta de retorno de la inversión, crear un ROI rectangular para la cuantificación de un animal enterond posicionarlo la imagen del ratón sobre para cubrir la punta de la nariz a la base de la cola del ratón. Utilice el mismo cuadro de tamaño para cada animal y cada punto de tiempo. Al mirar el retorno de la inversión para el cerebro, utilizar un ROI circular de la misma manera, el posicionamiento por encima de la región de la cabeza del ratón en la imagen. </li><li> Si se desea, ajustar las imágenes a aparecer en el mismo espectro de una representación visual de la infección. Nota: El software utilizado aquí genera un mapa de colores para cada imagen se ajusta automáticamente para el recuento mínimo y máximo de fotones. Para permitir la comparación visual entre una serie de imágenes, la escala de color debe estar ajustado a los mismos valores para todas las imágenes. <ol><li> El uso de la imagen de mayor bioluminiscencia y la pestaña &#39;Ajuste de imagen &quot;en la paleta de herramientas, seleccione una escala logarítmica y un mínimo de la escala de color adecuado y la máxima (a determinar empíricamente). Aplicar esta escala para todas las imágenes a lo largo del experimento. </li></ol></li></ol>

<ol>
	<li>Brun, R., Blum, J., Chappuis, F., Burri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis.">Human African trypanosomiasis.</a> Lancet. 375 (9709), 148-159 (2010).</li><li>Rodgers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosomiasis+and+the+brain.">Trypanosomiasis and the brain.</a> Parasitology. 137 (14), 1995-2006 (2010).</li><li>Barrett, M. P., Boykin, D. W., Brun, R., Tidwell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis:+pharmacological+re-engagement+with+a+neglected+disease.">Human African trypanosomiasis: pharmacological re-engagement with a neglected disease.</a> Br. J. Pharmacol. 152 (8), 1155-1171 (2007).</li><li>Barrett, M. P., Vincent, I. M., Burchmore, R. J. S., Kazibwe, A. J. N., Matovu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+in+human+African+trypanosomiasis.">Drug resistance in human African trypanosomiasis.</a> Future Microbiol. 6 (9), 1037-1047 (2011).</li><li>Jennings, F. W., Whitelaw, D. D., Urquhart, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+duration+of+infection+with+Trypanosoma+brucei+in+mice+and+the+efficacy+of+chemotherapy.">The relationship between duration of infection with Trypanosoma brucei in mice and the efficacy of chemotherapy.</a> Parasitology. 75 (2), 143-153 (1977).</li><li>Andreu, N., Zelmer, A., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biophotonic+imaging+for+studies+of+infectious+disease.">Noninvasive biophotonic imaging for studies of infectious disease.</a> FEMS Microbiol. Rev. 35 (2), 360-394 (2011).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc. Am. Thorac. Soc. 2 (6), 537-540 (2005).</li><li>Burrell-Saward, H., Rodgers, J., Bradley, B., Croft, S. L., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+reproducible+in+vivo+imaging+mouse+model+for+evaluation+of+drugs+against+late-stage+human+African+trypanosomiasis.">A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis.</a> J. Antimicrob. Chemother. 70 (2), 510-517 (2015).</li><li>McLatchie, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+in+vivo+imaging+of+Trypanosoma+brucei+expressing+'red-shifted'+luciferase.">Highly sensitive in vivo imaging of Trypanosoma brucei expressing 'red-shifted' luciferase.</a> PLoS Negl. Trop. Dis. 7 (11), e2571 (2013).</li><li>Jennings, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis:+potential+therapeutic+benefits+of+an+alternative+suramin+and+melarsoprol+regimen.">Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen.</a> Parasitol. Int. 51 (4), 381-388 (2002).</li><li>Wiles, S., Robertson, B. D., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+monitoring+of+in+vivo+colonization+and+clearance+dynamics+by+light-emitting+bacteria.">Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria.</a> Methods Mol. Biol. 574, 137-153 (2009).</li><li>Rodgers, J., Bradley, B., Kennedy, P. G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+chemotherapy+with+a+substance+P+receptor+antagonist+(aprepitant)+and+melarsoprol+in+a+mouse+model+of+human+African+trypanosomiasis.">Combination chemotherapy with a substance P receptor antagonist (aprepitant) and melarsoprol in a mouse model of human African trypanosomiasis.</a> Parasitol. Int. 56 (4), 321-324 (2007).</li><li>Kamerkar, S., Davis, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+on+the+brain:+understanding+host-pathogen+interactions+in+chronic+CNS+infection.">Toxoplasma on the brain: understanding host-pathogen interactions in chronic CNS infection.</a> J. Parasitol. Res. 2012, 589295 (2012).</li><li>Franke-Fayard, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+malaria+parasite+sequestration:+CD36+is+the+major+receptor,+but+cerebral+pathology+is+unlinked+to+sequestration.">Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (32), 11468-11473 (2005).</li><li>Hitziger, N., Dellacasa, I., Albiger, B., Barragan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+of+Toxoplasma+gondii+to+immunoprivileged+organs+and+role+of+Toll/interleukin-1+receptor+signalling+for+host+resistance+assessed+by+in+vivo+bioluminescence+imaging.">Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging.</a> Cell. Microbiol. 7 (6), 837-848 (2005).</li><li>Lewis, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+chronic+Trypanosoma+cruzi+infections+reveals+tissue-specific+parasite+dynamics+and+heart+disease+in+the+absence+of+locally+persistent+infection.">Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.</a> Cell. Microbiol. 16 (9), 1285-1300 (2014).</li></ol>

The authors have nothing to disclose.

El desarrollo de un T. bioluminiscente segundo. brucei cepa GVR35 permite la visualización de una infección por tripanosoma de principios a la etapa tardía. Modelos de infección anteriores eran incapaces de detectar la fase de retraso, cuando los parásitos están en el cerebro, en tiempo real desde la microscopía de frotis de sangre, y requieren el sacrificio y la eliminación de los cerebros de los ratones infectados para determinar la carga parasitaria 12. La bioluminiscencia reduce ...

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.

<table><tbody><tr><td>PBS</td><td>Sigma, UK</td><td>P4417</td><td>tablets pH 7.4</td></tr><tr><td>Glucose</td><td>Sigma, UK</td><td>G8270</td><td>99.5% (molecular) grade</td></tr><tr><td>Ammonium chloride</td><td>Sigma, UK</td><td>A9434</td><td>99.5% (molecular) grade</td></tr><tr><td>Heparin (lithium salt)</td><td>Sigma, UK</td><td>H0878</td><td></td></tr><tr><td>Hi-FCS</td><td>Gibco, Life Technologies, UK</td><td>10500-064</td><td>500 ml</td></tr><tr><td>DPBS</td><td>Sigma, UK</td><td>D4031</td><td>Sterile filtered</td></tr><tr><td>Mr. Frosty</td><td>Nalgene, UK</td><td></td><td></td></tr><tr><td>Giemsa</td><td>Sigma, UK</td><td>G5637</td><td></td></tr><tr><td>D-Luciferin</td><td>Perkin Elmer, UK</td><td></td><td></td></tr><tr><td>Sigma, UK</td><td>115144-35-9</td><td></td><td></td></tr><tr><td>Diminazene aceturate</td><td>Sigma, UK</td><td>D7770</td><td>Analytical grade</td></tr><tr><td>IVIS Lumina II</td><td>Perkin Elmer, UK</td><td></td><td>other bioimagers available &lt;em&gt;e.g.&lt;/em&gt; from Bruker, Kodak</td></tr><tr><td>Living Image v. 4.2</td><td>Perkin Elmer, UK</td><td></td><td>proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td></tr><tr><td>1 ml Syringe</td><td>Fisher Scientific, UK</td><td>10142104</td><td></td></tr><tr><td>20 ml Syringe</td><td>Fisher Scientific, UK</td><td>10743785</td><td></td></tr><tr><td>25 G Needles</td><td>Greiner Bio-one</td><td>N2516</td><td></td></tr><tr><td>21 G Needles</td><td>Greiner Bio-one</td><td>N2138</td><td></td></tr><tr><td>Twin-frosted microscope slide</td><td>VWR, UK</td><td>631-0117</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tube</td><td>StarLab, UK</td><td>I1415-1000</td><td></td></tr><tr><td>7 ml Bijou tube</td><td>StarLab, UK</td><td>E1412-0710</td><td></td></tr><tr><td>Mouse restrainer</td><td>Sigma, UK</td><td>Z756903</td><td>our restrainer was made in-house, this is a similar model</td></tr></tbody></table>

bioluminescence imaging to detect late stage infection of african trypanosomiasis

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.

Este protocolo demuestra cómo seguir la progresión de la enfermedad después de la infección de ratones con T. segundo. brucei, un modelo para la tripanosomiasis africana humana. La Figura 1 muestra la línea de tiempo del protocolo experimental, lo que demuestra el calendario de tratamiento de imágenes y pasos. La figura 2 muestra un campo típico de vista en un frotis de sangre teñido con Giemsa fija que se utiliza para cuantificar la par...

Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

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.

Imágenes de bioluminiscencia para detectar la infección en fase tardía de la tripanosomiasis africana

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 ...

bioluminescence-imaging-to-detect-late-stage-infection-african

Research

JoVE Journal

Immunology and Infection

7.7K vistas.  London School of Hygiene & Tropical Diseases. 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.

Video: Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

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.

Imágenes in vivo de los cultivos transgénicos parásitos Leishmania en un host en vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Investigación

Inmunología e Infección

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

Supervisión efectiva de laboratorio integral y costo del VIH / SIDA: un modelo a seguir de África

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

Bioluminiscencia de imágenes de la actividad NADPH oxidasa en diferentes modelos animales

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

En Vivo Imaging Systems (IVIS) La detección de un virus invasor Neuro-encefálico

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

De Alto Rendimiento de genes de marcado en las<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Programa de IA para la detección y clasificación de tripanosomiasis

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

Autor destacado: Detección de especies de tripanosomas impulsada por IA a partir de imágenes microscópicas 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

Identificación automática superior de parásitos tripanosomas mediante el uso de un modelo híbrido de aprendizaje profundo

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...

Bioingeniería

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Imagen de bioluminiscencia dual de la progresión tumoral y la angiogénesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

Imágenes de bioluminiscencia para detectar la infección en fase tardía de la tripanosomiasis africana

Resumen

Explorar más videos

Capítulos en este video

Imágenes in vivo de los cultivos transgénicos parásitos Leishmania en un host en vivo

Supervisión efectiva de laboratorio integral y costo del VIH / SIDA: un modelo a seguir de África

Bioluminiscencia de imágenes de la actividad NADPH oxidasa en diferentes modelos animales

En Vivo Imaging Systems (IVIS) La detección de un virus invasor Neuro-encefálico

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

De Alto Rendimiento de genes de marcado en las

Identificación automática superior de parásitos tripanosomas mediante el uso de un modelo híbrido de aprendizaje profundo

Identificación automática superior de parásitos tripanosomas mediante el uso de un modelo híbrido de aprendizaje profundo

Identificación automática superior de parásitos tripanosomas mediante el uso de un modelo híbrido de aprendizaje profundo

Imagen de bioluminiscencia dual de la progresión tumoral y la angiogénesis