Abstract The aim of this study was to describe the initial oral microbiota and how delivery mode and feeding practices impact its diversity in 0-2-month-old infants. This was a cross-sectional study that consisted of one collection of saliva samples from 0-2-month infants at baseline. Ten pairs of mothers and infants were selected. Medical health history, pregnancy, birth, feeding practices (breastfeeding or milk formula), and infant health status was obtained. Pooled microbial samples were obtained from the oral surfaces using a sterile cotton swab. Infants did not receive any breast milk before sampling. After collection, each swab was analyzed through microbiological culture-based procedures, using selective mediums. Cultures were analyzed for the presence of Streptococci, Lactobacillus, Staphylococcus, Enterobacterium , and Candida albicans . Twenty percent of the samples were serially diluted (10-2) to assess the number of bacteria expressed as CFU. Bacillota was the leading phylogenetic group in the infant’s pooled microbial sample. The most prevalent genera were Streptococcus, Lactobacillus , and Staphylococcus . Two participants had a positive growth of Candida albicans . The association between genus group, type of delivery, and feeding practices was not statistically significant (p > 0.05). Lactobacillus genus was frequently present in the cesarean delivery group but with slightly higher counts in a vaginal delivery study subject. Exclusively breastfed infants showed presence of Streptococcus, Lactobacillus, Staphylococcus . The oral microbiome in infants (0-2 month-old) is highly heterogeneous and dynamic. Microbiota composition seems to be impacted by mode of delivery, with slight differences among groups. Breastmilk appears as an essential factor in maintaining the oral microbiome’s stability and diversity.

Abstract The aim of this multicenter study was to explore the early-life sugar consumption and dietary practices in Latin America as well as to investigate the association between breastfeeding duration and the age at which foods and beverages with added sugars are introduced. A cross-sectional study was conducted with 805 1- to 3-year-old children from 10 Latin American countries, as a complementary study to the Research Observatory for Dental Caries of the Latin American Region (OICAL). A Food Frequency Questionnaire previously tested in different countries was applied to children’s mothers and data on breastfeeding and age at introduction of sugary foods and beverages was collected. Statistical analysis included the Kruskal-Wallis test and Poisson regression with robust variance, with the calculation of crude and adjusted mean ratios (MR) and 95% of confidence intervals (CI). The average age at introduction of sugary foods and beverages was 10.1 months (95%CI 9.7–10.4) and 9.6 (95%CI 9.2–9.9) months, respectively, with a significant variation between countries (p < 0.001). The average daily frequency of sugary foods-beverages was 3.3 times per day (95%CI 3.1–3.5) and varied significantly between countries (p = 0.004). Breastfeeding duration of over six months was associated with an increase in the age of introduction of sweet drinks (16%; MR 1.16; 95%CI 1.05–1.28) and foods (21%; MR 1.21; 95%CI 1.10–1.33). In conclusion, most children from vulnerable settings in Latin America start consuming sugary products in the first year of life and a high frequency of consumption was reported through early childhood. Additionally, breastfeeding contributes to a delay in the introduction of sugary products.

Abstract: This research aims to provide updated information on caries experience and associated risk factors in children 6-12 years old. A cross-sectional and descriptive study design was carried out with a non-probabilistic, convenient sample of 209 children male and female. Clinical examinations were performed by calibrated dental students following WHO detection criteria. Caries indices dmft and DMFT were calculated. Caries Risk Assessment data was collected using an adapted CAMBRA instrument; following the International Caries Care guidelines. Descriptive statistics were performed to analyze the results and Chi-square test, Contingency Coefficient (C) and Corrected Typified Residues were calculated to determine the association between variables. 58% of the total population had dental caries lesions in its more severe stages (cavitation) and 42% were apparently healthy (AHS) without any cavitated lesions. The mean dmft index was 1.34 ± 1.93, and the mean DMFT index was 0.63 ± 1.22. Lesion severity remained between 1-2 teeth affected on both dentitions. A statistically significant association (p = 0.035) between the health condition and toothbrushing was stablished with a degree of dependence of C = 0.144. A positive standardized residual of 2.1 was evident for schoolchildren that experience caries lesion that never brush their teeth and AHS that brushed their teeth more than once. No association (p = 0.081) was found between health condition and intake of sugary snacks and beverages. A severe dental caries experience with a statistically significant association between the health condition and toothbrushing with fluoridated toothpaste 1450 ppm > 1 a day and a positive correlation in schoolchildren that experience caries lesion that never brush their teeth.

Abstract Caries management at the lesion level is dependent on the lesion activity, the presence of a cavitation (either cleanable or non-cleanable), and lesion depth as evaluated via radiographic examination. A variety of non-invasive, micro-invasive, and minimally invasive treatment (with or without restoration) options are available for primary and permanent teeth. Non-invasive strategies include oral hygiene instructions, dietary counseling, and personal as well as professional use of fluoridated products that reduce demineralization and increase re-mineralization. Micro-invasive procedures include the use of occlusal resin sealants and resin infiltrants, while minimally invasive strategies comprise those related to selective removal of caries tissues and placement of restorations. Deep caries management includes indirect pulp capping, while exposed pulp may be treated using direct pulp capping and partial or complete pulpotomy. The aim of the present study was to review available evidence on recommended preventive and restorative strategies for caries lesions in Latin American/Caribbean countries, and subsequently develop evidence-based recommendations for treatment options that take into consideration material availability, emphasize ways to adapt available treatments to the local context, and suggest ways in which dentists and health systems can adopt these treatments.

ABSTRACT Bluetongue has been listed as a notifiable disease by the World Organization for Animal Health (OIE). Over the past two decades, the bluetongue virus has caused severe outbreaks in domestic and wild ruminants in several countries outside the range of 40°N and 35°S due to the migration of Culicoides spp. at increasing latitudes where ruminants are susceptible. Climate change, driven by global warming, could contribute to the spread of the virus beyond those limits , mainly through the creation of more suitable conditions for the propagation and reproduction of vectors. This review provides a summary of some previously compiled data of the different BTV serotypes that are present in the Americas and the Caribbean region, specified by serological techniques or viral isolation, as well as vector species involved in disease transmission.

RESUMEN La lengua azul (LA) es una enfermedad que se encuentra en la lista de enfermedades notificables por la Organización Mundial de Sanidad Animal (OIE). En las últimas dos décadas, el virus de la lengua azul (VLA) ha ocasionado severos brotes en rumiantes domésticos y silvestres en diversos países ubicados fuera del rango de 40°N y 35°S, debido a la migración de Culicoides spp. a latitudes cada vez mayores donde los rumiantes son susceptibles. El cambio climático, impulsado por el calentamiento global, podría contribuir a la propagación del virus sobre esos límites, principalmente mediante la creación de condiciones más adecuadas para la propagación y reproducción de vectores. Esta revisión brinda un resumen sobre algunos datos previamente compilados de los diferentes serotipos del VLA que están presentes en la región de las Américas y el Caribe, determinados por técnicas serológicas o aislamiento viral, así como las especies de vectores involucrados en la transmisión de la enfermedad.

ABSTRACT Bovine herpesvirus type 1 (BoHV-1) infects the respiratory and genital tracts of cattle and several species of ruminants. The infection can result in different diseases such as infectious bovine rhinotracheitis, infectious pustular vulvovaginitis in females and infectious pustular balanoposthitis in males, with a severe economic impact on livestock production. The infection causes respiratory symptoms, conjunctivitis, abortions, enteritis, and fatal systemic infection in newborn calves. One of the most important characteristics of bovine herpesvirus type 1 is the establishment of a latent infection that can be reactivated under stress conditions or by treatment with high doses of corticosteroids. The present work was aimed at developing an SYBR Green I Based Real-Time RT-PCR assay for the detection of bovine herpesvirus type 1, by amplifying a fragment of the viral thymidine kinase gene. The parameters of the critical components of the PCR reaction as well as the thermodynamic conditions were established. Dissociation curve analysis showed that the amplification curves were specific (Tm = 93°C ± 1°C) and that there were no unspecific amplifications. High levels of specificity, repeatability and sensitivity were obtained, with a detection limit of 100.5 TCID50/mL. Diagnostic sensitivity was assessed on the basis of nasal exudates from experimental breeding animals, reactivation of latent infection and experimental dairy farms. The SYBR Green I Based Real-Time RT-PCR assay proved to be more sensitive for the diagnosis of BoHV-1 than the endpoint PCR assay and virus isolation.

RESUMEN El herpesvirus bovino tipo 1 (BoHV-1) infecta los tractos respiratorio y genital del ganado bovino y varias especies de rumiantes. La infección puede resultar en diferentes enfermedades como rinotraqueitis infecciosa bovina, vulvovaginitis pustular infecciosa en hembras y balanopostitis pustular infecciosa en machos, con un severo impacto económico en la producción ganadera. La infección produce diferentes cuadros clínicos como respiratorios, conjuntivitis, abortos, enteritis e infección sistémica fatal en terneros recién nacidos. Una de las características más importante del herpesvirus bovino tipo 1 es el establecimiento de una infección latente que puede ser reactivada bajo condiciones de estrés o por tratamientos con altas dosis de corticosteroides. El presente trabajo tuvo como objetivo el desarrollo de un ensayo de PCR en tiempo real basado en SYBR-Green I para la detección de herpesvirus bovino 1, mediante la amplificación de un fragmento del gen de la timidina quinasa viral. Se establecieron los parámetros de los componentes críticos de la reacción de PCR, así como de las condiciones termodinámicas. Mediante el análisis de la curva de disociación se verificó que las curvas de amplificación fueron específicas (Tm = 93°C ± 1°C) y que no hubo amplificaciones inespecíficas. Se obtuvieron altos niveles de especificidad, repetibilidad y sensibilidad, con un límite de detección de 100.5 DICT50/mL. Se evaluó la sensibilidad diagnóstica a partir de exudados nasales procedentes de animales de una reproducción experimental, de reactivación de la infección latente y de vaquerías experimentales. El ensayo de PCR en tiempo real con SYBR-Green I demostró ser más sensible para el diagnóstico de BoHV-1, que el ensayo de PCR en punto final y el aislamiento viral.

ABSTRACT Equine influenza is an acute respiratory infection of horses, donkeys, mules and zebras. Nowadays, the reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR (rRT-PCR) assays are being widely used in diagnostic laboratories as a more sensitive alternative to virus isolation for the detection of equine influenza virus. The purpose of this study was to standardize an rRT-PCR, based on SYBR Green-I coupled with melting curve analysis for the detection of equine influenza virus (gene M) and the typing of hemagglutinin gene (HA). Sensitivity and specificity of the assay (gene M) were evaluated. In terms of the matrix gene copy number, the detection limit was 4.5 gene copies/μl of in vitro transcribed RNA. The linear range obtained for the transcript in the assay genered a typical standard curve from 107 until 100 gene copies/µl in terms of RNA copy number. The correlation coefficient (R2) was 0.99 for the RNA copies detected in nuclease free water and the error of standard curve was 0.0139. The rRT-PCR assay showed to be specific for equine influenza virus. No specific amplification curves were obtained with any of the other viruses tested such as equine viral arteritis, equine herpesvirus-1 and equine herpesvirus-4. The assay showed amplification and specific melting curves when the second set of primers against HA gene was evaluated. Thus, the proposed SYBR Green-I based rRT-PCR assay for the detection of equine influenza virus can be used in case of a possible emergency of this agent in Cuba.

RESUMEN La influenza equina es una infección respiratoria aguda de los caballos, burros, mulas y cebras. Actualmente, los ensayos de reverso transcripción acoplados a la reacción en cadena de la polimerasa (RT-PCR) y RT-PCR en tiempo real (rRT-PCR) están siendo ampliamente usados en los laboratorios de diagnóstico, como una alternativa más sensible al aislamiento viral, para la detección del virus de influenza equina. El objetivo de este estudio fue estandarizar un ensayo de rRT-PCR, basado en SYBR Green-I acoplado a análisis de curvas de disociación para la detección del virus de influenza equina (gen M) y la tipificación del gen de la hemaglutinina (HA). Se evaluaron la sensibilidad y la especificidad del ensayo (gen M). En términos de números de copias del gen de la matriz, el límite de detección fue de 4,5 copias del gen/μL del ARN transcripto in vitro. El rango lineal obtenido para el transcripto en el ensayo generó una curva estándar desde 107 hasta 100 copias del gen/µL en término de número de copias de ARN. El coeficiente de correlación (R2) fue de 0,99 para las copias de ARN detectadas en agua libre de nucleasas. El error de la curva estándar fue de 0,0139. El ensayo de rRT-PCR mostró ser específico para el virus de influenza equina. No se obtuvieron curvas de amplificación específicas con los otros virus evaluados, como son arteritis viral equina, herpesvirus equino-1 y herpesvirus equino-4. El ensayo mostró amplificación y curvas de disociación específicas cuando se evaluó la segunda pareja de cebadores contra el gen de la hemaglutinina. Así, el ensayo propuesto de rRT-PCR, basado en SYBR Green-I para la detección del virus de influenza equina, puede ser usado en caso de una posible emergencia de este agente en Cuba.

ABSTRACT The diagnosis and control of economically important viral diseases have progressed significantly in the last decade due to the application of polymerase chain reaction (PCR) technologies, as well as various applications thereof such as the PCR reaction based on the fluorescence detection in real time (rPCR), the loop-mediated isothermal amplification (LAMP) and the sequencing techniques based on nucleic acids. These approaches allow the amplification, simultaneous quantification and sequence analysis of nucleic acids, combining high speed with sensitivity and specificity, the benefits related to the conventional or endpoint PCR assays, a high dynamic range, a reduced cross contamination, the ability to be scaled, and the precise quantification of the target, allowing the determination of the viral load.

RESUMEN El diagnóstico y control de las enfermedades virales de importancia económica ha progresado de forma notable en la última década, gracias a la aplicación de las tecnologías de reacción en cadena de la polimerasa (PCR) y a las diferentes aplicaciones de la misma, como son la reacción de PCR basadas en la detección de fluorescencia en tiempo real (rPCR), la amplificación isotérmica mediada por bucle (LAMP) y las técnicas basadas en la secuenciación de ácidos nucleicos. Estos enfoques permiten la amplificación, la cuantificación simultánea y el análisis de secuencias de ácidos nucleicos, donde se combinan rapidez con elevadas sensibilidad y especificidad; sus beneficios con relación a los ensayos de PCR convencional o de punto final incluyen, además de un elevado rango dinámico, reducido riesgo de contaminación cruzada, capacidad para ser escalados y cuantificación precisa de la diana, lo que permite la determinación de la carga viral.

Avian leucosis/sarcoma virus (ALSV) provokes a variety of transmissible benign and malignant tumors affecting birds. Chickens are affected by six subgroups of ALSV designed as A, B, C, D, E and J, this last one is that of the most recent world dissemination. They were the first transmissible neoplastic diseases caused by viruses shown in any species 100 years ago, and they have consequently been studied extensively as models for the role of viruses in cancer by biomedical scientists. By the 1920s, they also became the major cause of mortality and economic losses in the poultry industry, and they have been studied by veterinary scientists for understanding and controlling them. Different detection methods for ALVS including enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunofluorescence assay (IFA), virus isolation, and routine PCR, have been developed. However, ELISA can detect only the group-specific antigen p27 and cannot differentiate between endogenous and exogenous viruses. Real-time PCR and immunofluorescence assays have been developed for both antigen detection and differentiation of endogenous and exogenous ALSV

El virus de la Leucosis/Sarcoma Aviar (ALSV) provoca una variedad de enfermedades tumorales benignas y malignas transmisibles que afectan a las aves. Los pollos se afectan por seis subgrupos de ALSV designados A, B, C, D, E y J; el último es el de más reciente difusión mundial. Estas fueron las primeras enfermedades neoplásicas transmisibles causadas por virus que se mostraron hace 100 años atrás y, por lo tanto, han sido ampliamente estudiadas por los científicos biomédicos como modelos para estudiar la relación de los virus con el cáncer. También se convirtieron, a partir del año 1920, en la principal causa de mortalidad y pérdidas económicas para la industria avícola, por lo que se han estudiado por los científicos veterinarios en busca de comprender y controlar la enfermedad que provocan. Diferentes métodos de detección de los virus de leucosis aviar se han desarrollado, entre los que se pueden mencionar los ensayos inmunoenzimáticos (ELISA), ensayo de inmunofluorescencia (IFA), el aislamiento viral, PCR en punto final y PCR en tiempo real. Sin embargo, los ELISA desarrollados pueden detectar solo el antígeno específico del grupo p27 y no pueden diferenciar entre virus endógenos o exógenos. Se han desarrollado ensayos de PCR en tiempo real y de inmunofluorescencia para la diferenciación de los ALSV endógenos y exógenos y detección de antígenos

Bluetongue (BT) is an insect-transmitted viral disease of ruminant species that primarily affects sheep. This disease is caused by a virus (BTV) of the genus Orbivirus within the family Reoviridae. Twenty-six distinct serotypes of the virus have been identified to date. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate BTV detection methods, which can assist in disease control. SYBR Green-based real-time reverse transcription-PCR (rRT-PCR) assay may be an ideal method to detect rapidly the BTV genome in the blood of animals. In this study, a rRT-PCR coupled to melting curve analysis was described and compared with the nested reverse transcription-polymerase chain reaction (nRT-PCR) reported in the OIE Manual. The specificity and sensitivity of both methods were evaluated using BTV-4 RNA extracted from tenfold serially diluted tissue culture medium virus and blood (starting from 10(4.5) TCID50/ml). The detection limit of each test in tissue culture medium was 10(0.5) TCID50/ml and in blood was 10(1.5) TCID50/ml (rRT-PCR) and 10(0.5) TCID50/ml (nRT-PCR). Melting curve analysis showed that the rRT-PCR yielded curves of amplification with specific melting curves (Tm = 84°C ± 0.5°C) and absence of non-specific amplifications. The diagnostic sensibility of clinical samples from a calf under controlled conditions was evaluated. These results showed that the SYBR Green I-based real-time PCR assay is rapid, sensitive, and equally specific in the diagnosis of BT in BTV-affected animals.

La lengua azul es una enfermedad viral de especies de rumiantes que se transmite por insectos, y que primariamente afecta a los carneros. La enfermedad es causada por un virus (BTV) que pertenece al género Orbivirus de la familia Reoviridae. Se han identificado 26 serotipos diferentes de este virus hasta la fecha. Debido a la rápida diseminación del virus y a la variabilidad de los signos clínicos, se necesita el desarrollo de métodos de detección de BTV rápidos y precisos, los cuales pueden ayudar en el control de la enfermedad. Un ensayo de RT-PCR en tiempo real (rRT-PCR), basado en SYBR Green-I, puede ser un método ideal para detectar rápidamente el genoma de BTV en la sangre de los animales. En este estudio se describe un análisis de curvas de disociación acoplado al rRT-PCR y se compara con el ensayo de RT-PCR anidado (nRT-PCR) que se reporta en el Manual de la OIE. La especificidad y la sensibilidad de ambos métodos se evaluaron usando ARN de BTV-4 extraído a partir de diluciones seriadas del virus en medio de cultivo de tejido y en sangre (comenzando a partir de 10(4.5) TCID50/ml). El límite de detección de cada ensayo en medio de cultivo de tejido fue de 10(0.5) TCID50/ml y en sangre fue de 10(1.5) TCID50/ml (rRT-PCR) y 10(0.5) TCID50/ml (nRT-PCR). Los análisis de curvas de disociación mostraron curvas de amplificación con curvas de disociación específicas (Tm = 84°C ± 0.5°C) y ausencia de amplificación no específica. La sensibilidad diagnóstica se evaluó a partir de muestras clínicas de un ternero bajo condiciones controladas. Estos resultados mostraron que el rRT-PCR, basado en SYBR Green-I, es un ensayo rápido, sensible e igualmente específico en el diagnóstico de animales afectados con BT.

Bluetongue is a non-contagious acute viral disease affecting ruminants. It is a disease of great clinical importance for the physical deterioration and the long convalescence that it causes and, from the economic point of view, for the colossal production losses and prevention and control expenses. It is highly feared in spite of being innocuous for man and of only medium mortality. Bluetongue virus spreads from animal to animal by biting insects of the genus Culicoides and this is the reason for the disease being more prevalent in the geographic areas where climate conditions are favorable for the insect development. The disease was described in detail in early 1900, and at present, is one of the main concerns in animal health, being studied in order to clarify its epidemiology and pathogenesis and to control its spreading. In this review, an attempt has been made to summarize some aspects of the disease related to its history, economic importance and etiological agent, the viral genome organization, the disease epidemiology and distribution, its clinical signs and lesions, as well as its diagnosis.

La Lengua azul es una enfermedad viral no contagiosa, aguda, que afecta a los rumiantes. Es una enfermedad de gran importancia clínica por el deterioro físico y la larga convalecencia que provoca y, desde el punto de vista económico, por las colosales pérdidas de producción y gastos de prevención y control. Es muy temida pese a ser inocua para el hombre y de solo mediana mortalidad. El virus se transmite entre los animales por picaduras de insectos del género Culicoides, lo que hace que su prevalencia sea mayor en las zonas geográficas donde el clima favorece el desarrollo de los mismos. Esta enfermedad se describió en detalle a principios del siglo XIX y aún hoy constituye una de las principales preocupaciones en la salud animal, por lo que se continúa estudiando para aclarar su epidemiología, patogenia y controlar así su diseminación. Esta revisión tiene como objetivo resumir algunos aspectos de la enfermedad relacionados con su historia, importancia económica, agente etiológico, organización del genoma viral, la epidemiología y la distribución, los signos clínicos y lesiones, así como el diagnóstico de la enfermedad.

Avian infectious bronchitis (AIB) is a viral disease worldwide distributed and it is considered one of the main causes of economic losses for poultry industry because of its characteristic of affecting development of broiler and laying hens. In Cuba, the current diagnosis is carried out by viral isolation and its identification by conventional methods is difficult. In order to identify the possible presence of avian infectious bonchitis virus in cuban isolates a reverse transcription-polymerase chain reaction assay (RT-PCR) specific for this virus was applied. Trachea-lung samples were taken as pooles of three and four birds after each necropsy, evaluating a total of eleven birds. The sequences of AIB virus were detected in two of three pools. In one of these pools the detection from clinical sample was also obtained, a very important aspect for its differentiation with serious diseases with a similar clinical course as Avian influenza and Newcastle disease. The RT-PCR showed a potential in order to be used in the identification of isolates from AIB virus, as well as in the direct examination of clinical samples opening the possibilities of its use in the quick, sensitive and specific detection of this agent in our country an essential aspect in the diagnosis and control of this viral infection.

La bronquitis infecciosa aviar (BIA) es una enfermedad viral ampliamente diseminada en el mundo y se considera una de las principales causas de pérdidas económicas significativas en la industria avícola, debido a que afecta el desarrollo de aves de engorde y ponedoras. En Cuba, el diagnóstico actual se realiza por aislamiento viral y es difícil su identificación por métodos convencionales. Para identificar la posible presencia del virus de la BIA en aislados cubanos se aplicó un ensayo de reverso transcripción acoplado a reacción en cadena de la polimerasa (RT-PCR) específico para este virus. Las muestras de tráquea-pulmón fueron tomadas como grupos de tres y cuatro aves después de la necropsia de cada una, evaluándose un total de once aves. Las secuencias del virus de la BIA fueron detectadas en dos de los tres grupos evaluados. En uno de estos grupos se logró además, la detección del virus a partir de la muestra clínica; aspecto importante para la diferenciación con enfermedades graves que cursan con un cuadro clínico similar como la Influenza aviar y la Enfermedad de Newcastle. La RT-PCR mostró potencial para ser usado en la identificación de aislados del virus de la BIA, así como en el examen directo a partir de muestras clínicas, lo que aporta posibilidades de su empleo en nuestro país para la detección rápida, sensible y específica de este agente, aspecto esencial en el diagnóstico y control de esta infección viral.

Avian influenza is a great threat for animal and human health, thus strengthening diagnostic systems against probable outbreaks is a priority all over the world. That is why detection is important by the use of molecular methods (RT-PCR and Real Time RT-PCR) due to their high speed, sensitivity and specificity allowing to take control measures in order to avoid the dissemination of the agent. In this work, the obtaining and evaluation of amplification and quantification positive controls for RT-PCR and Real Time RT-PCR (RRT-PCR) have been described. They were obtained from the cloning of two PCR products, one of them corresponding to a region of the subtype H5 hemagglutinin gene and the other to the matrix gene (M) for Influenza type A. The primers were selected keeping in mind that the PCR products included the corresponding regions of H5 and influenza type A primer couples reported by RT-PCR and RRT-PCR of OIE/ FAO reference laboratories for the diagnosis of this disease. From an RNA extracted from H5N2 strain (kindly donated by the OIE reference laboratory for avian influenza), the cDNA used as a mold for the amplification with the primers J3/J1C for H5 subtype and M2 M+25 for Influenza type A, recommended by reference laboratories, was obtained. A clone from which a fragment of the size expected was amplified, was selected as a positive control in the detection assays of nucleic acids for the H5 subtype and influenza A type. Also, the H5 plasmid was evaluated for RRT-PCR and a standard curve with a Ct of 15 was obtained. The amplification control obtained for H5 subtype detection will allow to standardize assays and evaluate possible negative falses for the quick detection of this subtype in Cuba; and in the case of the positive control for influenza type A it will be used for quantification in RRT-PCR assays.

La influenza aviar (IA) constituye una gran amenaza para la salud animal y humana por lo que es una prioridad a nivel mundial el fortalecimiento de los sistemas de diagnóstico frente a probables brotes, de ahí la importancia de la detección por métodos moleculares (RT-PCR y Real time RT-PCR) por su elevada rapidez, sensibilidad y especificidad de estos que permiten tomar las medidas de control para evitar la diseminación del agente. En este trabajo se describe la obtención y evaluación de controles positivos de amplificación y cuantificación para RT-PCR y Real time RT-PCR (RRT-PCR) obtenidos a partir del clonaje de dos productos de PCR, uno correspondiente a una región del gen de la hemaglutinina del subtipo H5 y el otro al gen de la matriz (M) para Influenza tipo A. Los cebadores se seleccionaron teniendo en cuenta que los productos de PCR incluyeran las regiones correspondientes de parejas de cebadores de H5 e influenza tipo A reportados tanto por RT-PCR y RRT-PCR de laboratorios de referencia de la OIE/FAO para el diagnóstico de esta enfermedad. De un RNA extraído a partir de la cepa H5N2 (gentilmente donado por el laboratorio de referencia para Influenza aviar de la OIE) se obtuvo el cDNA que se utilizó como molde para la amplificación con los cebadores J3/J1C para H5 y M2/M+25 para Influenza tipo A, recomendados por laboratorios de referencia. Se seleccionó un clón a partir del cual se logró amplificar un fragmento de la talla esperada, que se ha utilizado como control positivo en los ensayos de detección de ácidos nucleicos para el subtipo H5 y para influenza tipo A. Además, el plásmido de H5 se evaluó por RRT-PCR y se obtuvo una curva estándar con un Ct de 15. El control de amplificación obtenido para la detección del subtipo H5 permitirá estandarizar los ensayos y evaluar posibles falsos negativos para la detección rápida de este subtipo en Cuba y en el caso del control positivo de influenza tipo A se podrá usar para cuantificación en ensayos de RRT-PCR.

Avian leukosis viruses (ALV) provoke a variety of trasmissible bening and malign tumoral diseases affecting birds. Chickens are affected by six subgroups of ALV designs A, B, C, D, E and J of more recent world dissemination. These viruses are potential contaminants of live vaccines used in poultry. In order to research the presence of DNA from ALVs as contaminants of viral commercial vaccines to be used in poultry, different Marek´s disease vaccines were screened by a reported polymerase chain reaction (PCR) assay designed to detect all subgroups of ALVs. DNA samples extracted from seven vaccines were submitted to PCR using primers for a conserved region of env gene of HPRS-103. ALV sequences were detected in seven samples (100%). The methodology employed proved to be useful for the detection of ALVs as contaminants of imported Marek´s disease vaccines. These data suggest a high occurrence of ALVs in commercial vaccines intended for poultry disease prevention.

Los virus de la leucosis aviar (ALV) provocan una variedad de enfermedades tumorales benignas y malignas transmisibles que afectan a las aves. Los pollos son afectados por seis subgrupos de ALV designados A, B, C, D, E y J de más reciente diseminación mundial. Estos virus son, además, potenciales contaminantes de vacunas vivas usadas en la avicultura. Para investigar la presencia de ADN de ALV como contaminante de vacunas virales comerciales usadas en la avicultura, monitoreamos diferentes vacunas de la enfermedad de Marek en un ensayo reportado por Reacción de la Cadena de la Polimerasa (PCR) diseñado para detectar todos los subgrupos del ALV. Las muestras de ADN extraídas de siete vacunas fueron evaluadas por PCR utilizando cebadores para una región conservada del gen de la envoltura (env) de HPRS-103. Las secuencias del ALV fueron detectadas en las siete muestras (100%). La metodología empleada resultó útil para la detección de ALV como contaminante de vacunas de Marek importadas. Nuestros datos sugieren una elevada presencia de ALV en vacunas comerciales destinadas a la prevención de enfermedad en la avicultura.