In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD-420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.Fil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Faverin, Claudia. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Boynak, Natalia. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vena, M. M.. Biogénesis-Bagó S. A.; ArgentinaFil: Zabal, O.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentin

Diagnostic ultrasound estimates of muscle mass and muscle quality discriminate between women with and without sarcopenia

Introduction: Age-related changes in muscle mass and muscle tissue composition contribute to diminished strength in older adults. The objectives of this study are to examine if an assessment method using mobile diagnostic ultrasound augments well-known determinants of lean body mass (LBM) to aid sarcopenia staging, and if a sonographic measure of muscle quality is associated with muscle performance.Methods: Twenty community-dwelling female subjects participated in the study (age = 43.4 ±20.9 years; BMI: 23.8, interquartile range: 8.5). Dual energy X-ray absorptiometry (DXA) and diagnostic ultrasound morphometry were used to estimate LBM. Muscle tissue quality was estimated via the echogenicity using grayscale histogram analysis. Peak force was measured with grip dynamometry and scaled for body size. Bivariate and multiple regression analyses were used to determine the association of the predictor variables with appendicular lean mass (aLM/ht2), and examine the relationship between scaled peak force values and muscle echogenicity. The sarcopenia LBM cut point value of 6.75 kg/m2 determined participant assignment into the Normal LBM and Low LBM subgroups.Results: The selected LBM predictor variables were body mass index (BMI), ultrasound morphometry, and age. Although BMI exhibited a significant positive relationship with aLM/ht2 (adj. R2 = .61, p \u3c .001), the strength of association improved with the addition of ultrasound morphometry and age as predictor variables (adj. R2 = .85, p \u3c .001). Scaled peak force was associated with age and echogenicity (adj. R2 = .53, p \u3c .001), but not LBM. The Low LBM subgroup of women (n = 10) had higher scaled peak force, lower BMI, and lower echogenicity values in comparison to the Normal LBM subgroup (n = 10; p \u3c .05).Conclusions: Diagnostic ultrasound morphometry values are associated with LBM, and improve the BMI predictive model for aLM/ht2 in women. In addition, ultrasound proxy measures of muscle quality are more strongly associated with strength than muscle mass within the study sample

Diagnostic ultrasound estimates of muscle mass and muscle quality discriminate between women with and without sarcopenia

Introduction: Age-related changes in muscle mass and muscle tissue composition contribute to diminished strength in older adults. The objectives of this study are to examine if an assessment method using mobile diagnostic ultrasound augments well-known determinants of lean body mass (LBM) to aid sarcopenia staging, and if a sonographic measure of muscle quality is associated with muscle performance.Methods: Twenty community-dwelling female subjects participated in the study (age = 43.4 ±20.9 years; BMI: 23.8, interquartile range: 8.5). Dual energy X-ray absorptiometry (DXA) and diagnostic ultrasound morphometry were used to estimate LBM. Muscle tissue quality was estimated via the echogenicity using grayscale histogram analysis. Peak force was measured with grip dynamometry and scaled for body size. Bivariate and multiple regression analyses were used to determine the association of the predictor variables with appendicular lean mass (aLM/ht2), and examine the relationship between scaled peak force values and muscle echogenicity. The sarcopenia LBM cut point value of 6.75 kg/m2 determined participant assignment into the Normal LBM and Low LBM subgroups.Results: The selected LBM predictor variables were body mass index (BMI), ultrasound morphometry, and age. Although BMI exhibited a significant positive relationship with aLM/ht2 (adj. R2 = .61, p \u3c .001), the strength of association improved with the addition of ultrasound morphometry and age as predictor variables (adj. R2 = .85, p \u3c .001). Scaled peak force was associated with age and echogenicity (adj. R2 = .53, p \u3c .001), but not LBM. The Low LBM subgroup of women (n = 10) had higher scaled peak force, lower BMI, and lower echogenicity values in comparison to the Normal LBM subgroup (n = 10; p \u3c .05).Conclusions: Diagnostic ultrasound morphometry values are associated with LBM, and improve the BMI predictive model for aLM/ht2 in women. In addition, ultrasound proxy measures of muscle quality are more strongly associated with strength than muscle mass within the study sample

Differential immunity as a factor influencing mussel hybrid zone structure

Interspecific hybridisation can alter fitness-related traits, including the response to pathogens, yet immunity is rarely investigated as a potential driver of hybrid zone dynamics, particularly in invertebrates. We investigated the immune response of mussels from a sympatric population at Croyde Bay, within the hybrid zone of Mytilus edulis and Mytilus galloprovincialis in Southwest England. The site is characterised by size-dependent variation in genotype frequencies, with a higher frequency of M. galloprovincialis alleles in large mussels, largely attributed to selective mortality in favour of the M. galloprovincialis genotype. To determine if differences in immune response may contribute to this size-dependent variation in genotype frequencies, we assessed the two pure species and their hybrids in their phagocytic abilities when subject to immune challenge as a measure of immunocompetence and measured the metabolic cost of mounting an antigen-stimulated immune response. Mussels identified as M. galloprovincialis had a greater immunocompetence response at a lower metabolic cost compared to mussels identified as M. edulis. Mussels identified as hybrids had intermediate values for both parameters, providing no evidence for heterosis but suggesting that increased susceptibility compared to M. galloprovincialis may be attributed to the M. edulis genotype. The results indicate phenotypic differences in the face of pathogenic infection, which may be a contributing factor to the differential mortality in favour of M. galloprovincialis, and the size-dependent variation in genotype frequencies associated with this contact zone. We propose that immunity may contribute to European mussel hybrid zone dynamics

Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin

Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.<br>Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus

Obtención y evaluación de cultivos primarios de origen epitelial para el aislamiento de los virus CAEV y ORFV

Trabajo presentado en la XXI Reunión científico-técnica Dr. Bernardo Jorge Carrillo : Asociación Argentina de Veterinarios de Laboratorio de Diagnóstico, celebrada en San Salvador de Jujuy (Argentina), del 6 al 8 de octubre de 2016En el diagnóstico confirmatorio de enfermedades virales se suelen emplear técnicas serológicas (ELISA e inmunodifusión en agar AGID) o moleculares (PCR, RT-PCR) para la detección de los agentes virales. Si bien, muchas veces los reactivos necesarios suelen ser caros y poco accesibles al evaluar un gran número de muestras. Además, en muestras biológicas donde la carga viral suele ser baja, las técnicas antes mencionadas suelen presentar limitaciones. La forma de confirmar la presencia del agente viral es obtener su aislamiento en cultivo de células, siguiendo la aparición del efecto citopático característico para cada uno de ellos. Sin embargo, es habitual que la cepas de campo no se adapten a su multiplicación in vitro en líneas celulares establecidas, por lo que muchas veces es necesario recurrir a los cultivos primarios. El objetivo de este trabajo consistió en la generación de un cultivo primario de origen epitelial ovino y caprino con el fin de obtener un sistema celular que permita el aislamiento de los virus de artritis y encefalitis caprina (CAEV) y el virus ORF (ORFV),agente causal del Ectima contagioso, a partir de muestras sospechosas.Peer reviewe

Topical Ganciclovir Reduces Viral Excretion in Mares With Equine Coital Exanthema

Equid alphaherpesvirus 3 (EHV-3) is the etiological agent of equine coital exanthema (ECE). Because no vaccines or antiviral therapies are available, prevention consists of clinical examination of mares and stallions before mating or semen collection and resting from breeding activities when lesions are present. However, this methodology does not identify subclinically infected animals. Ganciclovir is the most potent compound known to reduce EHV-3 replication. This study aimed to evaluate the efficacy of topical ganciclovir application to reduce EHV-3 replication in experimentally infected mares. A pilot study, after a double-blind completely randomized design, was carried out. Twenty mares were randomly divided into five groups (three treated with ganciclovir with different regimen of doses, one treated with a placebo, and one nontreated). Mares were experimentally infected with EHV-3 on day 0. Rectal temperature, clinical signs, and lesions were recorded. Daily perineal and vaginal swabs were evaluated by quantitative polymerase chain reaction for virus detection. The antibody response was assessed by a virus neutralization test in serum samples collected weekly. Mares experimentally infected with EHV-3 and treated with ganciclovir twice a day for 13 days showed reduced levels and duration of viral excretion and less severe lesions. The viral excretion period was reduced from 18 to nine days compared with the untreated groups. We concluded that ganciclovir had an antiviral effect on EHV-3 replication when topically administered in mares showing clinical signs of ECE. Further trials should be performed to optimize the dose of the antiviral for a definitive formulation. © 202