Infección experimental en ovinos con el virus de leucosis bovina: dosis mínima de células BLV-FLK y virus libre de células, y actividad neutralizante de anticuerpos naturales / Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies

Abstract Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Resumen El virus de la leucosis bovina (bovine leukemia virus [BLV]) es un importante agente patógeno del ganado que causa importantes pérdidas económicas en todo el mundo, especialmente en los rodeos lecheros. El uso de modelos animales proporciona información valiosa sobre la patogénesis de las infecciones virales. Se realizaron infecciones experimentales en ovejas usando sangre de bovinos infectados con BLV, clones moleculares de BLV infecciosos o células derivadas de tumores. La línea celular Fetal Lamb Kidney, persistentemente infectada con el BLV (FLK-BLV), es uno de los cultivos a largo plazo más utilizados para la producción permanente de virus. Las células FLK-BLV o las partículas virales obtenidas del sobrenadante del cultivo libre de células podrían usarse como fuente de provirus o de virus para infectar experimentalmente ovejas. En este trabajo, nuestro objetivo fue determinar la cantidad mínima de células FLK-BLV o de sobrenadante libre de células que contiene BLV necesaria para producir infección en ovejas. También evaluamos la cantidad de anticuerpos bovinos anti-BLV necesaria para neutralizar la infección. Observamos que las dos ovejas inoculadas experimentalmente con 5000 células FLK-BLV se infectaron, y que una de las dos ovejas que recibieron 500 células FLK-BLV se infectó. Ninguno de los animales inoculados con 50 células FLK-BLV mostró evidencia de infección. El sobrenadante FLK-BLV libre de células demostró ser infectivo en ovejas hasta la dilución 1:1000. Los anticuerpos BLV específicos mostraron actividad neutralizante, ya que ninguna de las ovejas se infectó. Por el contrario, los animales que recibieron un suero BLV negativo mostraron signos de infección por BLV. Estos resultados contribuyen a la optimización de un bioensayo en ovejas útil para caracterizar la infección por BLV.

Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies.

Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model.

AIM OF THE STUDY: To determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM). MATERIAL AND METHODS: Canine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105 CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing. RESULTS: Bacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5×10(-3). All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10×WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants. CONCLUSIONS: Intracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge.

El factor de crecimiento del endotelio vascular hepático en la infección murina por Schistosoma mansoni / The endothelial growth factor vascular infection in murine liver by Schistosoma mansoni

Los antígenos secretados por los huevos de S. mansoni inducen la proliferación de células endoteliales in vivo, así como la producción del Factor de Crecimiento del Endotelio Vascular (VEGF), sugiriendo que la patogénesis en la esquistosomiasis se relaciona con eventos angiogénicos. Se evaluó la expresión del VEGF, como una medida de angiogénesis estimulada por los huevos y los gusanos de S. mansoni, en la infección Bisexual (BIS) y los gusanos adulto (infección UNI) en el hígado de ratones Balb/c, antes y después del tratamiento con PZQ. Los resultados indican que tanto la infección UNI como BIS son capaces de estimular la producción de VEGF en tejido hepático, lo que explica la vascularizacón anómala durante este cuadro infeccioso. Este proceso se acompaña con la presencia de un elevado número de infiltrados leucocitarios en los sitios donde se observa lesión tisular; la producción de VEFG remite tras 48 de tratamiento con PZQ. Estos resultados indican que la producción anómala de VEGF junto con la intensa respuesta pro-inflamatoria asociada no solo a la actividad de VEGF sino también a los infiltrados leucocitarios observados en el tejido hepático, causada tanto por los huevos secretados como por las formas adultas de S. mansoni son los mecanismos que subyacen a las lesiones granulomatosas observadas durante el curso de la esquistosomiasis, pudiendo al menos revertirse el incremento en la vascularización mediante el uso de PZQ.

The antigens secreted by eggs of S. mansoni induce the proliferation of endothelial cells in vivo, as well as the production of Factor Vascular Endothelial Growth factor (VEGF), suggesting that the pathogenesis of schistosomiasis relations with angiogenic events. We evaluated the expression of VEGF, as a measure of angiogenesis stimulated by the eggs and worms S. mansoni in infection bisexual (BIS) and adult worms ( UNI infection ) in the liver of BALB / c mice before and after treatment with PZQ. These results indicate that abnormal production of VEGF with intense pro-inflammatory response not only associated with the activity of VEGF but also leukocyte infiltrates observed in the liver tissue caused by both secreted and eggs by adult forms S. mansoni are the mechanisms underlying granulomatous lesions observed during the course of schistosomiasis, and can be reversed at least the increased vascularization by using PZQ .

Dosis mínima infectante de la fase micelial de Histoplasma capsulatum var. capsulatum, en ratones Balb/c / Minimal infective dose of the mycelium phase of Histoplasma capsulatum var. capsulatum in Balb/c mice

El objetivo fue determinar la dosis mínima infectante (DMI) de Histoplasma capsulatum mediante inoculación de la fase micelial en ratones Balb/c, con el propósito de aportar alternativas útiles para el futuro uso de inóculos fúngicos que faciliten la reproducción de la enfermedad. Se prepararon inóculos de 50, 75 y 94% de transmitancia (T) provenientes de la mezcla de suspensiones estandarizadas de cinco aislamientos del hongo y a cada inóculo se le determinaron las unidades formadoras de colonias/mL (UFC/mL). Se inyectaron, intraperitonealmente, 100 µL de cada inóculo a tres grupos de 10 ratones, identificados como G2, G3 y G4, respectivamente. Se incluyó otro grupo como control sano (G1) y se observaron durante 15 días. La enfermedad se comprobó mediante la observación de los ratones, estudios histopatológicos y micológicos. La DMI fue de 50% T (7,5 x 10(4) UFC/mL), seleccionada en función de la presencia de hepatoesplenomegalia, nódulos en hígado y/o bazo y el porcentaje de recuperación del hongo en los subcultivos. Los resultados demostraron la reproducción de la enfermedad, con ausencia de muertes en el transcurso de 15 días de observación.

The objective was to determine the Histoplasma capsulatum minimal infective dose (MID) by the inoculation of the mycelium phase in Balb/c mice, with the purpose of contributing useful alternatives for the future application of fungal inoculums which facilitate the reproduction of the disease. Several inoculums with 50, 75 and 94% transmittance (T) were prepared with a mixture of standardized suspensions from five isolations of the fungus, determining colony forming units/ml (CFU/mL) for each inoculum. Three groups of 10 mice each, identified as G2, G3 and G4, were injected intraperitoneally with 100 µl of each inoculum. Another group (G1) was included as healthy control. All mice were observed during 15 days. The disease was determined by observation of the animals, and by histopathological and mycological examinations. The MID was 50% T (17.5 x 10(4) CFU/mL ) selected on basis of the presence of hepatosplenomegaly, nodules in liver and/or spleen, and the percentage of recovery of fungi from subcultures. The results showed the reproduction of the disease in absence of deaths during the 15-day observation period.

Estudio comparativo de la susceptibilidad de cuatro especies del genero Rhodnius a la infección experimental con Trypanosoma cruzi / Comparative study of susceptibility of four species of genera Rhodnius to experimental infection with Trypanosoma cruzi in Venezuela

Con el objetivo de comparar su susceptibilidad, se infectaron experimentalmente con Trypanosoma cruzi especímenes de Rhodnius prolixus, Rhodnius robustus, Rhodnius neivai y Rhodnius neglectus. El análisis de varianza de Kruskall-Wallis con los datos agrupados por especie y por estadio reveló diferencias estadísticamente significativas entre las especies en cuanto al volumen de sangre ingerida, volumen de orina producida y número de parásitos desarrollados en orina y heces para cada estadio. Posteriormente la prueba de comparación múltiple de Mínima Diferencia Significativa demostró que R. prolixus ingirió el volumen más elevado de sangre mientras que R.neivai produjo el volumen de orina más elevado, seguido por R. robustus, R. prolixus y R. neglectus,en ese orden. Asimismo, R. neivai mostró el promedio más elevado de parásitos tanto en orina como en heces mientras que R. robustus y R. neglectus produjeron significativamente menos parásitos en orina y heces respectivamente. Las cuatro especies estudiadas son capaces de infectarse, multiplicar y excretar el parásito en su forma infectante, en todos los estadios

In order to compare their susceptibility to Trypanosoma cruzi specimens of Rhodnius prolixus, R.robustus, R. neivai and R. neglectus were experimentallyinfected with this parasite. Statistically significantdifferences among the species in the volume of bloodingested, volume of urine produced and number ofparasites in urine and grounds developed in each stage,were detected with a non parametric Kruskall-Wallisanalysis of variance, with the data grouped in speciesand instars. The Low Significative Difference posterioritest, shows that R. prolixus ingested the highest bloodvolume while R. neivai produced the highest urinevolume, followed by R. robustus, R. prolixus and R.neglectus. Likewise, R. neivai showed the highest averageof parasites in urine and feces, while R. robustus and R.neglectus showed significantly less parasites in urineand feces respectively. The four studied species are ableto infect, to multiply and to excrete the parasite in their infective form in all instars stages