Análisis de expresión de genes de Mycobacterium tuberculosis durante la infección a macrófagos

Los factores bacterianos responsables de la patogenicidad de M. tuberculosis no han sido plenamente identificados. En este trabajo, se analizó el efecto citotóxico producido por esta bacteria sobre macrófagos humanos en cultivo, así como la expresión de algunos genes bacterianos que pudieran estar involucrados en el proceso de infección. Monocapas en cultivo de la línea celular de monocitos humanos (THP-1) diferenciada a macrófagos por inducción con forbol-miristrato-acetato (PMA) se infectaron con M. tuberculosis cepas CDC1551, H37Rv y DR689, con una multiplicidad de infección (MDI) de 10:1, 1:1 y 0.1:1 (bacterias:macrófagos). El efecto citotóxico se observó como aglomeración celular y áreas de lisis. El daño dependió de la concentración del inóculo y del tiempo de incubación. En infecciones realizadas con una MDI de 10:1, se observó destrucción total de los cultivos a 24 h de incubación. Utilizando MDI menores se observó un daño menor en el cultivo celular y aglomeración de células en varias zonas, los cuales no se presentaron en los cultivos testigo. Las monocapas celulares infectadas con una MDI de 1:1 y 0.1:1 conservaron parcialmente su integridad a las 72 h de incubación. Para cuantificar el efecto citotóxico producido por M. tuberculosis, se desarrolló un método colorimétrico basado en la característica particular de M. tuberculosis de no absorber el cristal violeta. De esta manera la perdida de células en el cultivo debido al efecto citotóxico se refleja cuantitativamente en las lecturas de absorbancia. En base a los resultados de citotoxicidad se seleccionó la MDI de 1:1 para realizar los estudios de expresión durante la infección. La extracción del RNA bacteriano se realizó de bacterias obtenidas a los 7 días de crecimiento en medio de cultivo Middlebrook 7H9, de bacterias cultivadas en medio RPMI completo o de bacterias infectando macrófagos a las 2, 6 y 24 h de incubación. La selección de genes para analizar su expresión se hizo tomando como base genes de M. tuberculosis reportados como probables factores de virulencia o genes sin función definida en M. tuberculosis pero con homología con factores de virulencia en otros patógenos. Los genes seleccionados para buscar su expresión se nombraron de acuerdo a la nomenclatura dada en http://www.sanger.ac.uk/Projects/M_tuberculosis/: MT0003, MT0024, MT0040, MT0259, MT0655, MT0876, MT1524, MT3917 (erp), MT2783 (sigB), MT2415 (plcB) y MT2414 (plcC). El testigo de expresión constitutiva fue el gene MT3989 (esat6), el cual se expresó en todas las condiciones analizadas. Como testigo de expresión inducida durante la infección se seleccionó el gene MT2416 (plcA), el cual se expresó de manera semejante a lo reportado previamente por otros autores. Todos los genes a excepción del MT0259 se expresaron en todas las condiciones, pero se observó variabilidad en la intensidad de las bandas lo que representa una posible expresión diferencial que debe establecerse mediante métodos cuantitativos. El gene MT0259 con función aparente de oxidoreductasa se expresó hasta las 24 h de incubación tanto en medio de cultivo como en células infectadas pero no mostró expresión determinada por RT-PCR a los 7 días de crecimiento en medio Middlebrook. Con estos hallazgos, se concluyó que M. tuberculosis tiene un efecto citotóxico sobre la monocapa de macrófagos cuantificable por la tinción con cristal violeta y que la bacteria expresa selectivamente ciertos genes dependiendo de las condiciones del medio de cultivo. Abstract M. tuberculosis virulence factors have not been completely identified. In this work, the cytotoxic effect produced by M. tuberculosis on human macrophages and the expression of specific bacterial genes during macrophage infection that may be involved in mycobacterial virulence was analysed. THP-1 cells monolayers differentiated to macrophages by phorbol myristate acetate (PMA) were infected with M. tuberculosis strains CDC1551, H37Rv and DR689 with a variable multiplicity of infection (MOI): 10:1, 1:1 and 0.1:1 (bacteria:macrophage) and incubated at various time points. Cytotoxicity was observed as areas of clearing and cell agglomeration and the effect was dependent on bacilli inoculum and incubation time. MOI of 10:1 produced a complete destruction of the cell monolayer after 24 h post-infection. Uninfected cultures kept their integrity throughout the experiment. Cell monolayers infected with MOI’s of 1:1 and 0.1:1 were partially disrupted after 72 h post-infection. In order to measure the cytotoxic effect, we developed an in vitro colorimetric assay based on M. tuberculosis inability to absorbe crystal violet dye, such that any lost of cells caused by M. tuberculosis in infected cultures would produce a change in absorbance values. Based on the cytotoxicity results, the MOI of 1:1 was selected to recover bacteria, isolate their RNA and perform the expression analysis. RNA was isolated from bacteria grown in Middlebrook 7H9 medium after 7 days or in RPMI medium at 2, 6 and 24 h, as well as RNA from bacteria infecting macrophages at the same time points. Selection of analysed genes was based on genes reported as probable virulence factors of M. tuberculosis or genes with undefined function in M. tuberculosis but having a nucleotide sequence homologous to virulence genes from others pathogens. Selected genes for this study were named according to the nomenclature given in http://www.sanger.ac.uk/Projects/M_tuberculosis/: MT0003, MT0024, MT0040, MT0259, MT0655, MT0876, MT1524, MT3917 (erp), MT2783 (sigB), MT2415 (plcB) and MT2414 (plcC). Gene MT3989 (esat6) was used as control of constitutive expression and gene MT2416 (plcA) as control of induced expression during infection. Gene MT0259 with a putative oxidoreductase activity did not show expression after 7 days of growth in 7H9 medium as determined by RT-PCR. But, it did express when cultivated in RPMI or during the macrophage infection at least till 24 h. All the rest of the genes analysed were expressed in all the studied conditions. Although, variability was observed on the intensity of bands, indicating a probable upregulation or dowregulation of expression, it is necessary to make quantitative analysis to get conclusive results. These findings indicate that M. tuberculosis has a cytotoxic effect on macrophage monolayers, which can be measured by crystal violet dye and that M. tuberculosis selectively express genes depending on culture conditions

Bluetongue in white-tailed deer (Odocoileus virginianus) in Northeastern Mexico

Abstract Bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) are two distinct viral hemorrhagic diseases of domestic and wild ruminants caused by members of the family Reoviridae and transmitted by Culicoides midges. These conditions have been recognized in Canada and the United States for many years, but not in Mexico. Although in Mexico there is serologic evidence of EHD virus (EHDV) and bluetongue virus (BTV) in domestic and wild ruminants, to our knowledge, there have never been reports of clinical illness or fatalities attributed to either of these viruses. Two free ranging white-tailed deer (Odocoileus virginianus) in two licensed hunting ranches in Northern Mexico near the Texas border died unexpectedly. Postmortem and microscopic examinations revealed hemorrhagic lesions compatible with viral hemorrhagic disease (Reoviridae: Orbivirus). Tissues from one animal tested positive by RT-PCR for BTV but negative for EHDV. To our knowledge, this is the first time in Mexico where deer dying with hemorrhagic lesions consistent with Bluetongue tested positive for BTV by PCR. Key words: hemorrhagic disease, white-tailed deer, Orbivirus, Mexico

Inseminação artificial e transferência de embriões em tempo fixo em bovinos de corte sob condições tropicais secas

Gestation percentage was assessed after fixed time embryo transfer (FTET) and artificial insemination (FTAI) programs as a means to evaluate its use to improve reproductive efficiency in beef herds in Tamaulipas, under dry tropic conditions. Two FTET programas were conducted the north part of the state, the first, at the ranch Puesta de Sol (Burgos), where 30 beef crossbred heifers were treated with CIDR plus estradiol benzoate and cipionate for FTET; the second was conducted at the ranch La India (Reynosa), where 95 beef crossbred heifers using the same treatment. Embryos from Beefmaster donors were produced at the Laboratorio FIV of UGRT and transferred fresh, to 23 heifers at Puesta de Sol and 77 heifers at La India, where 8 and 32 gestations were obtained, for 35 and 42%, respectively, for a total gestation rate of 40%. The FTAI program was conducted at the ranch San Isidro (Guémez), where 133 registered Red Brangus (50 cows) and Charolaise (88 cows) were treated with the protocol described for FTET, plus an injection of 330 IU of eCG to nursing cows and cows in poor body condition. A total gestation rate of 41% was obtained, gestation rate was greater for Red Brangus (50%) than for Charolaise (35%) cows; partial gestation rates were 34 and 24% and 23 and 16%, respectively for both programs and breeds. Differences for both FTET and FTAI programs were not significant. Results obtained in this study allow recommending the use of such biotechnological tools to improve reproductive efficiency in beef herds in Tamaulipas.Se determinó el porcentaje de gestación (PG) para transferencia de embriones (TETF) e  inseminación artificial (IATF) a tiempo fijo, para bovinos de carne, Tamaulipas, bajo condiciones de trópico seco. Se realizaron dos programas de TETF en el norte de Tamaulipas, el primero en el Rancho Puesta de Sol, Burgos, donde se trataron 30 vaquillas con CIDR + benzoato de estradiol (BE) y cipionato de estradiol (ECP) al retiro del CIDR; el segundo programa se realizó en el Rancho La India (Reynosa), donde se sincronizaron 95 vaquillas cruzadas, con el mismo tratamiento. Se realizaron 23 transferencias en Puesta de Sol y 77 transferencias en La India, con embriones de FIV, de la raza Beefmaster, los cuales fueron producidos en el Laboratorio FIV, de la UGRT. Se obtuvieron 8 y 32 gestaciones, para 35 y 42%, respectivamente, para Puesta de Sol y la India, para un total de 40% de gestación total. El programa de IATF se realizó en el Rancho San Isidro (Guémez), utilizando 133 vacas de registro, 50 Brangus Rojo y 88 Charolais, se utilizó el mismo tratamiento que para TETF, además, las vacas paridas y en condición corporal menor de 2.5, se trataron con 330 UI de eCG, al retiro del CIDR, las vacas se inseminaron entre 55 y 60 horas post-retiro del CIDR; las vacas vacías se re-sincronizaron, utilizando el mismo protocolo. Se obtuvo PG de 41%, y 50% en Brangus Rojo y 35% en Charolais; los parciales para PG fueron de 34 y 24% y de 23 y 16%, respectivamente para ambos programas y ambas razas. Las diferencias no fueron significativas en TETF ni para IATF. Se concluye que se pueden implementar programas de TETF e IATF, para mejorar la eficiencia reproductiva de los hatos en Tamaulipas.A porcentagem de gestação (PG) foi determinada para transferência de embriões (TETF) e inseminação artificial (AITF) em tempo fixo, para bovinos de corte, Tamaulipas, sob condições tropicais secas. Dois programas de TETF foram realizados no norte de Tamaulipas, o primeiro em Rancho Puesta de Sol, Burgos, onde 30 novilhas foram tratadas com CIDR mais benzoato de estradiol (BE) e cipionato de estradiol (ECP) após a retirada do CIDR; O segundo programa foi realizado no Rancho La India (Reynosa), onde foram sincronizadas 95 novilhas cruzadas, com o mesmo tratamento. Foram realizadas 23 transferências em Puesta de Sol e 77 transferências em La India, com embriões FIV da raça Beefmaster, produzidos no Laboratório de FIV da UGRT. Obtiveram-se 8 e 32 gestações, para 35 e 42%, respectivamente, para Puesta de Sol e Índia, para um total de 40% da gestação total. O programa IATF foi realizado no Rancho San Isidro (Guémez), usando 133 vacas registradas, 50 Red Brangus e 88 Charolês, o mesmo tratamento que para TETF foi usado, além disso, vacas paridas e com condição corporal inferior a 2,5, foram tratadas com 330 UI de eCG, na retirada do CIDR, as vacas foram inseminadas entre 55 e 60 horas após a retirada do CIDR; vacas vazias foram ressincronizadas, usando o mesmo protocolo. Obteve-se PG de 41%, sendo 50% no Brangus Vermelho e 35% no Charolês; as parciais para PG foram 34 e 24% e 23 e 16%, respectivamente para ambos os programas e ambas as raças. As diferenças não foram significativas no TETF nem no IATF. Conclui-se que os programas TETF e IATF podem ser implementados para melhorar a eficiência reprodutiva dos rebanhos em Tamaulipas

Activity of Semi-Synthetic Mulinanes against MDR, Pre-XDR, and XDR Strains of Mycobacterium tuberculosis

Tuberculosis causes more than 1.2 million deaths each year. Worldwide, it is the first cause of death by a single infectious agent. The emergence of drug-resistant strains has limited pharmacological treatment of the disease and today, new drugs are urgently needed. Semi-synthetic mulinanes have previously shown important activity against multidrug-resistant (MDR) Mycobacterium tuberculosis. In this investigation, a new set of semi-synthetic mulinanes were synthetized, characterized, and evaluated for their in vitro activity against three drug-resistant clinical isolates of M. tuberculosis: MDR, pre-extensively Drug-Resistant (pre-XDR), and extensively Drug-Resistant (XDR), and against the drug-susceptible laboratory reference strain H37Rv. Derivative 1a showed the best anti-TB activity (minimum inhibitory concentration [MIC] = 5.4 µM) against the susceptible strain and was twice as potent (MIC = 2.7 µM) on the MDR, pre-XDR, and XDR strains and also possessed a bactericidal effect. Derivative 1a was also tested for its anti-TB activity in mice infected with the MDR strain. In this case, 1a produced a significant reduction of pulmonary bacilli loads, six times lower than the control, when tested at 0.2536 mg/Kg. In addition, 1a demonstrated an adjuvant effect by shortening second-line chemotherapy. Finally, the selectivity index of >15.64 shown by 1a when tested on Vero cells makes this derivative an important candidate for future studies in the development of novel antitubercular agents