Sequence-based epitope mapping of high pathogenicity avian influenza H5 clade 2.3.4.4b in Latin America.

High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.

Sequence-Based Antigenic Analyses of H1 Swine Influenza A Viruses from Colombia (2008-2021) Reveals Temporal and Geographical Antigenic Variations.

Swine influenza is a respiratory disease that affects the pork industry and is a public health threat. It is caused by type A influenza virus (FLUAV), which continuously undergoes genetic and antigenic variations. A large amount of information regarding FLUAV in pigs is available worldwide, but it is limited in Latin America. The HA sequences of H1 subtype FLUAV-positive samples obtained from pigs in Colombia between 2008-2021 were analyzed using sequence-based antigenic cartography and N-Glycosylation analyses. Of the 12 predicted global antigenic groups, Colombia contained five: four corresponding to pandemic strains and one to the classical swine H1N1 clade. Circulation of these clusters was observed in some regions during specific years. Ca2 was the immunodominant epitope among Colombian viruses. The counts of N-Glycosylation motifs were associated with the antigenic cluster ranging from three to five. The results show for the first time the existence of antigenic diversity of FLUAV in Colombia and highlight the impact of spatial and temporal factors on this diversity. This study provides information about FLUAV variability in pigs under natural conditions in the absence of vaccination and emphasizes the need for surveillance of its phylogenetic and antigenic characteristics.

New Insights into Avian Infectious Bronchitis Virus in Colombia from Whole-Genome Analysis.

Infectious Bronchitis (IB) is a respiratory disease caused by a highly variable Gammacoronavirus, which generates a negative impact on poultry health worldwide. GI-11 and GI-16 lineages have been identified in South America based on Infectious Bronchitis virus (IBV) partial S1 sequences. However, full genome sequence information is limited. In this study we report, for the first time, the whole-genome sequence of IBV from Colombia. Seven IBV isolates obtained during 2012 and 2013 from farms with respiratory disease compatible with IB were selected and the complete genome sequence was obtained by NGS. According to S1 sequence phylogenetic analysis, six isolates belong to lineage GI-1 and one to lineage GVI-1. When whole genome was analyzed, five isolates were related to the vaccine strain Ma5 2016 and two showed mosaic genomes. Results from complete S1 sequence analysis provides further support for the hypothesis that GVI-1, considered a geographically confined lineage in Asia, could have originated in Colombia. Complete genome information reported in this research allow a deeper understanding of the phylogenetic evolution of variants and the recombination events between strains that are circulating worldwide, contributing to the knowledge of coronavirus in Latin America and the world.

Zooming in on the molecular characteristics of swine influenza virus circulating in Colombia before and after the H1N1pdm09 virus.

Influenza is one of the most critical viral agents involved in the respiratory disease complex affecting swine production systems worldwide. Despite the absence of vaccination against swine influenza virus in Colombia, the serologic reactivity to classic H1N1 and H3N2 subtypes reported since 1971 indicates the virus has been circulating in the country's swine population for several decades. However, successful isolation and sequencing of field virus from pigs was nonexistent until 2008, when H1N1 classical influenza virus was identified. One year later, due to the emergence of the influenza A (H1N1) pdm09 virus, responsible for the first global flu pandemic of the 21st century, it was introduced in the country. Therefore, to understand the impact of the introduction of the H1N1pdm09 virus in Colombia on the complexity and dynamics of influenza viruses previously present in the swine population, we carried out a study aiming to characterize circulating viruses genetically and establish possible reassortment events that might have happened between endemic influenza viruses before and after the introduction of the pandemic virus. A phylogenetic analysis of ten swine influenza virus isolates from porcine samples obtained between 2008 and 2015 was conducted. As a result, a displacement of the classical swine influenza virus with the pdmH1N1 virus in the swine population was confirmed. Once established, the pandemic subtype exhibited phylogenetic segregation based on a geographic pattern in all the evaluated segments. The evidence presents reassortment events with classic viruses in one of the first H1N1pdm09 isolates. Thus, this study demonstrates complex competition dynamics and variations in Colombian swine viruses through Drift and Shift.

Characterization and evaluation of Colombian propolis on the intestinal integrity of broilers.

Nutritional additives such as propolis seek to improve intestinal health as an alternative to the global ban on in-feed antibiotics used as growth promoters (AGP). The objective of this study was to evaluate the effect of propolis supplementation in diet of broilers. Four hundred and fifty straight-run Ross 308 AP broilers were fed with a basal diet (BD) throughout the whole experimental period. Birds were randomly distributed into 5 groups at d 14: negative control without antibiotics nor propolis (AGP-), positive control 500 ppm of Zinc Bacitracin as growth promoter (AGP+), and 3 groups supplemented with 150, 300, and 450 ppm of propolis. Every group included 6 replicates of 15 birds each. Propolis concentration was increased from d 22 to 42, in experimental groups to 300, 600, and 900 ppm of propolis, and 10% of raw soybean was included as a challenge in all groups during the same period. Analysis of productive parameters, intestinal morphometry, and relative quantification of genes associated with epithelial integrity by qPCR were performed at 21 and 42 d. The groups with the greatest weights were those that consumed diets including 150 (21 d) and 900 ppm (42 d) of propolis compared with all treatments. The lowest score of ISI was found at 300 (21 d) and 600 ppm (42 d). A lower degree of injury in digestive system was seen with the inclusion of 300 ppm (21 d) and 900 ppm (42 d). Up-regulation of zonula occludens-1 (ZO-1) was observed in jejunum of broilers supplemented with 150 and 300 ppm at 21 d. Up-regulation of ZO-1 and TGF-ß was also evidenced in ileum at all propolis inclusion levels at 42-day-old compared to AGP+ and AGP-. The beneficial effects were evidenced at inclusion levels of 150 ppm in the starter and 900 ppm in the finisher. According to the results, the Colombian propolis inclusion can improve productive performance, physiological parameters, and gene expression associated with intestinal integrity.

Molecular Evidence of Orthomyxovirus Presence in Colombian Neotropical Bats.

The Orthomyxoviridae family includes the genera Influenzavirus, Isavirus, Quaranjavirus, and Thogotovirus. In turn, Influenzavirus can be classified into four types: α, ß, γ, and δ (Formerly A, B, C, and D), from which Alphainfluenzavirus (AIV) has the broadest host range, including birds, mammals, reptiles, and amphibians. Additionally, AIV has shown global epidemiological relevance owing to its pandemic potential. The epidemiological relevance of Chiropteran due to its multiple functional characteristics makes them ideal reservoirs for many viral agents. Recently, new influenza-like subtypes have been reported in Neotropical bats, but little is known about the relevance of bats as natural reservoirs of influenza viruses. Therefore, the current study aimed to determine the presence of AIV and new influenza-like subtypes in South American bats. For a better understanding of the drivers and interactions between AIV and bats, we used molecular assays with different gene targets (i.e., M, NP, and PB1) to identify AIV in New World bats. A housekeeping gene (CytB) PCR was used to check for nucleic acid preservation and to demonstrate the bat-origin of the samples. A total of 87 free-living bats belonging to 25 different species of the families Phyllostomidae and Vespertilionidae were collected in Casanare, Colombia. As a result, this study found seven AIV-positive bat species, three of them reported for the first time as AIV prone hosts. Neither of the AIV-like analyzed samples were positive for H17N10/H18/N11 subtypes. Although additional information is needed, the presence of a completely new or divergent AIV subtype in neotropical bats cannot be discarded. Collectively, the results presented here expand the epidemiological knowledge and distribution of AIV in neotropical free-ranging bats and emphasize the need to continue studying these viruses to establish the role they could play as a threat to animal and public health.

Key Factors That Enable the Pandemic Potential of RNA Viruses and Inter-Species Transmission: A Systematic Review.

Viruses play a primary role as etiological agents of pandemics worldwide. Although there has been progress in identifying the molecular features of both viruses and hosts, the extent of the impact these and other factors have that contribute to interspecies transmission and their relationship with the emergence of diseases are poorly understood. The objective of this review was to analyze the factors related to the characteristics inherent to RNA viruses accountable for pandemics in the last 20 years which facilitate infection, promote interspecies jump, and assist in the generation of zoonotic infections with pandemic potential. The search resulted in 48 research articles that met the inclusion criteria. Changes adopted by RNA viruses are influenced by environmental and host-related factors, which define their ability to adapt. Population density, host distribution, migration patterns, and the loss of natural habitats, among others, have been associated as factors in the virus-host interaction. This review also included a critical analysis of the Latin American context, considering its diverse and unique social, cultural, and biodiversity characteristics. The scarcity of scientific information is striking, thus, a call to local institutions and governments to invest more resources and efforts to the study of these factors in the region is key.

Influenza needs an approach as a "one health" problem in Colombia / Influenza requiere un manejo bajo la perspectiva de "una salud" en colombia

RESUMEN La influenza es una infección viral de importancia y distribución mundial, cuyo agente causal es el Alfainfluenzavirus o influenza virus tipo A (IAV), el cual se caracteriza por poseer un genoma de tipo ssRNA segmentado, aspecto que le confiere una alta variabilidad y capacidad recombinante. Esto, sumado al amplio rango de huéspedes susceptibles y la posibilidad de transmisión entre especies, se constituye en un reto tanto para la salud humana como para la animal. El IAV es capaz de infectar una amplia variedad de huéspedes, incluyendo múltiples especies de aves y mamíferos, tanto domésticas como salvajes y al humano, así como a reptiles y anfibios, entre otros. Dentro de los Alphainfluenzavirus se reconocen 16 subtipos de Hemaglutinina (HA) y nueve de Neuraminidasa (NA), siendo su principal reservorio las aves silvestres acuáticas. Adicionalmente, se han reconocido dos nuevos subtipos en murciélagos (H17-18 y N10-11), los cuales se han denominado Influenza-like virus. Teniendo en cuenta lo anterior y conocedores de la riqueza en biodiversidad que posee Colombia, país en el que está demostrada la circulación del virus en cerdos y en humanos y hay resultados preliminares de la presencia de Orthomyxovirus en murciélagos, es imperativo estudiar y conocer los IAV circulantes en el medio, establecer factores de riesgo y analizar el efecto que han tenido y seguirán teniendo las condiciones asociadas al cambio climático, los factores sociodemográficos y el papel de diferentes especies en la ecología de este agente viral. Todo lo anterior bajo el contexto de "Una Salud" en la infección por IAV.

ABSTRACT Influenza is an important viral disease of worldwide distribution. It is caused by the Alfainfluenzavirus or influenza virus type A (IAV). A segmented ssRNA genome in the influenza viruses confers high variability and reassortment capability to the virus. That and the broad range of susceptible hosts, along with the possibility of inter-species transmission, represents a challenge to human and animal health. The IAV is able to infect a large variety of hosts such as several wild and domestic avian and mammalian species, including humans, as well as reptiles and amphibians, among others. There are 16 hemagglutinin (HA) and nine neuraminidase (NA) subtypes recognized until know, whose main reservoir are the wild aquatic birds. In addition, two new subtypes (H17-18 and N10-11) have been recognized in bats, and these have been designated as influenza-like viruses. Taking this into account and knowing the richness of biodiversity in Colombia, there is an imperative need to study and to know about the IAV circulating in the field in order to establish risk factors and to analyze the past, the current and the future effect that climate change, sociodemographic factors and the role that different species could play in the eco-biology of this viral agent. This should be considered under the one health concept of influenza virus infection as a whole, considering the fact that Colombia is a country in which the circulation of IAV has been demonstrated in the swine and human population and there are preliminary results of the presence of Orthomyxovirus in bats.

Identification of genogroups of Infectious Bursal Disease Virus in poultry farms at Colombia / Identificación de genogrupos del virus de la enfermedad de gumboro en granjas avícolas en colombia

RESUMEN El virus de la enfermedad de Gumboro (IBDV) es un avibirnavirus con genoma dsARN que presenta altas tasas de mutación y recombinación. A pesar del efecto inmunosupresor en aves y la frecuencia con que ocurre la infección por este agente en el país son pocos los estudios que caracterizan los cuadros clínicos y se desconoce cuáles son los genogrupos circulantes. Esta investigación tuvo como objetivo determinar la frecuencia de lesiones histopatológicas en órganos del sistema inmune e identificar los genogrupos del IBDV en aves comerciales de Colombia. Para determinar la frecuencia de presentación de lesiones en órganos del sistema inmune se analizaron 381 casos clínicos de las bases de datos del Laboratorio de Patología Aviar (LPA) de la Universidad Nacional de Colombia, sede Bogotá (periodo 2016-2018). Asimismo, se secuenciaron los productos de RT-PCR del gen que codifica para la proteína viral VP2 provenientes de 35 muestras de bursas de Fabricio. Como resultado se encontró evidencia de lesiones microscópicas compatibles con procesos de inmunodepresión en órganos del sistema inmune (bursa de Fabricio, timo, bazo y médula ósea) en el 25 % (97) de los casos analizados y se identificaron los genogrupos 1, 2 y 4 en la siguiente proporción: genogrupo 1-69 % (virus clásicos), genogrupo 2-25 % (variantes) y genogrupo 4-6 % (identificado en Suramérica). Estos hallazgos demuestran la presencia de lesiones en órganos del sistema inmune y la existencia de los genogrupos 1, 2 y 3 del IBDV circulando en aves comerciales en Colombia. Esta es la primera investigación en el país con este sistema de clasificación que permite evidenciar con mayor precisión los cambios en el genoma del IBDV. Lo anterior señala la necesidad de continuar con este tipo de estudios para tener una mejor comprensión de la infección en campo y orientar el diseño e implementación de estrategias de control.

ABSTRACT Infectious Bursal Disease Virus (IBDV) is an avibirnavirus with a dsRNA genome, which has a high mutation and recombination rates. Despite the immunosuppressive effect in poultry and the frequency of infections by this agent, few studies in Colombia that characterize the clinical signs and identify the circulating genogroups have been reported. This study aimed to determine the frequency of histopathological lesions in immune organs and to identify IBDV genogroups in poultry from Colombia. In this way, the Laboratorio de Patología Aviar (LPA) databases from the Universidad Nacional de Colombia (period 2016-2018) were analyzed in order to identify the frequency of lesions present in immune organs. 35 samples of the bursa of Fabricius, positive by RT-PCR, were sequenced to the gene that codes for the VP2 protein. 97 (25 %) cases showed microscopic lesions in the immune organs. The genogroups identified and the frequencies were: genogroup 1-69 % (classical viruses), genogroup 2-25 % (antigenic variants), and genogroup 4-6 % (identified in South America). These findings demonstrate the lesions of immune organs and the presence of different genogroups circulating in commercial birds in Colombia. This indicates the need to continue with these studies in order to have a better understanding of the infection in the field and to guide the design and implementation of control strategies.

Detection of equine herpesvirus 1 and 4 and its association with latency-associated transcripts in naturally infected horses in Colombia / Detección de los herpesvirus equinos 1 y 4 y su relación con transcriptos asociados a la latencia en caballos infectados naturalmente en Colombia

ABSTRACT Objective. Determine the presence of antibodies and viral genomes of EHV-1 and EHV-4, as well as to detect the presence of latency associated transcripts (LATs) in a selected population of Colombian horses. Materials and methods. Serum samples, submandibular lymph nodes and trigeminal ganglion were obtained from 50 horses and analyzed. Sera were evaluated for the presence of antibodies against EHV-1 and EHV-4 while tissues were initially evaluated for the presence of viral genome by nPCR. Finally, samples were used for the detection of LATs through RT-PCR. Results. In general, 6/50 samples showed antibodies to EHV-1 and 44/50 were positive for EHV-4. As for viral genome detection, 10/50 samples were positive for EHV-1 and 30/50 were positive for EHV-4; in addition, 22/35 horses positive for EHV DNA were positive for LATs. The use of these tests led to eight possible combinations of results. Conclusions. The evidence used shows that horses can have simple viral infection, co-infections with both viruses, latency due to the presence of LATs and the simultaneous presence of LATs and viral genome replication at a given time. It contributes to the understanding of the behavior of the disease in Colombia and calls attention to the importance of implementing complementary diagnoses to the serology for the control of these viruses.

RESUMEN Objetivo. Determinar presencia de anticuerpos y genoma viral de EHV-1 y EHV-4, como también detectar la presencia de transcriptos asociados a latencia (LATs) en una población seleccionada de caballos colombianos. Materiales y métodos. Muestras de suero, nódulos linfáticos submandibulares y ganglio trigémino se obtuvieron de 50 caballos y fueron analizadas. Los sueros se evaluaron para la presencia de anticuerpos contra EHV-1 y EHV-4 mientras que los tejidos se evaluaron inicialmente para la presencia de genoma viral por nPCR. Finalmente, las muestras se emplearon para la detección de LATs a través de RT-PCR. Resultados. En general, 6/50 muestras mostraron anticuerpos para EHV-1 y 44/50 fueron positivos para EHV-4. En cuanto a la detección del genoma viral, 10/50 muestras fueron positivas para EHV-1 y 30/50 fueron positivas para EHV-4; además, 22/35 caballos positivos para DNA de EHV fueron positivos para LATs. El empleo de estas pruebas llevó a ocho posibles combinaciones de resultados. Conclusiones. Se confirma la presencia de estos virus en la población equina colombiana. Las pruebas empleadas demuestran que los caballos pueden tener infección viral simple, co-infecciones con ambos virus, estado de latencia debido a la presencia de los LATs y presencia simultánea de LATs y de replicación de genoma viral en un momento dado. Se aporta al entendimiento del comportamiento de la enfermedad en Colombia y se llama la atención sobre la importancia de implementar diagnósticos complementarios a la serología para el control de estos virus.

Molecular characterization of Marek's disease virus in a poultry layer farm from Colombia.

Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.

Dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts, and the effect on their litters / Dinámica de la infección por el circovirus porcino tipo 2 y títulos de anticuerpos neutralizantes en las cerdas de reemplazo subclinicamente infectadas, y el efecto en sus camadas / Dinâmica da infeção pelo circovirus porcino tipo 2 e títulos de anticorpos neutralizantes em porcas nulíparas subclínicamente infectadas e efeito em sua leitegada

Background: porcine circovirus type 2 (PCV2) is associated with reproductive disease in newly populated herds and in replacement breeding stock from new sources and is almost exclusively reported in gilts. Objective: the main purpose of this study was to assess the dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts and the effect on their piglets. Methods: the study was conducted with 40 gilts selected at random from four breeding herds. Blood samples, nasal and vaginal swabs were obtained from the gilts at arrival, acclimatization, farrowing, and one day after farrowing. Colostrum samples were collected immediately after parturition and one day after farrowing. Blood, nasal swab, or tissue samples were collected from four piglets prior to suckling. All serums were analyzed by virus neutralization test (VNT) to establish the presence of antibodies. All samples were subjected to SYBER Green real-time PCR assay to detect PCV2 DNA. Results: high levels of viremia and viral load of PCV2 in nasal and vaginal swabs were found in healthy gilts at arriving, confirming the introduction of infected animals into the farms. In addition, most gilts were positive for PCV2 DNA in serum, nasal and vaginal swabs at farrowing. PCV2 shedding was also observed in nasal and vaginal fluids and colostrum even in presence of serum neutralizing antibodies (NA). Subclinically infected dams had detectable viremia, developed anti-PCV2 antibodies, and there was PCV2 DNA in tissue samples of their born alive and healthy piglets. PCV2a and PCV2b genotypes were confirmed in PCV2 subclinical infection in both dams and piglets in utero. Conclusion: replacement gilts can be infected with PCV2 before entering the farm and continuous exposure seems to occur horizontally in acclimatization and gestation units or before farrowing. Exposure and infection during gestation may result in infected but apparently healthy piglets.

Antecedentes: el circovirus porcino tipo 2 (PCV2) es asociado con casos de falla reproductivas en granjas recién pobladas, en granjas de cría para cerdas jóvenes y casi exclusivamente en cerdas de reemplazo. Los signos clínicos descritos son: incremento en los abortos durante la segunda y tercera etapa de la gestación, fetos momificados, mortinatos y el nacimiento de lechones débiles no viables. Objetivo: el principal propósito de este estudio fue evaluar la dinámica de la infección por el circovirus porcino tipo 2 y títulos de anticuerpos neutralizantes en las cerdas de reemplazo subclinicamente infectadas y el efecto en su camada. Métodos: este estudio se realizó con 40 cerdas de reemplazo seleccionadas al azar en cuatro granjas porcinas de cría. De cada animal se colectaron muestras de sangre, hisopados nasales y vaginales al ingresar a la explotación, durante la cuarentena, en el momento del parto y un día post-parto. Igualmente, se colectaron muestras de calostro al terminar el parto y un día post-parto. De cuatro lechones neonatos, se colectaron muestras de sangre, hisopado nasal y tejidos antes de consumir calostro. Todos los sueros fueron analizados mediante la técnica de sero-neutralización para detectar anticuerpos anti-PCV2 y todas las muestras se analizaron por una técnica SYBER Green en tiempo real para detectar el ADN viral. Resultados: la detección de un alto nivel de viremia y la demostración de la eliminación viral en hisopados nasales y vaginales permitió demostrar la introducción a las granjas de cerdas de reemplazo infectadas, aparentemente sanas. Igualmente, el suero y los hisopados nasales y vaginales fueron positivos por PCR SYBER Green en la mayoría de las hembras al parto. Se demostró eliminación viral en fluidos nasales, vaginales y en calostro en presencia de anticuerpos séricos neutralizantes. La infección de las cerdas se manifestó en viremia, en el desarrollo de anticuerpos frente al PCV2 y en la presencia del ADN viral en los tejidos de lechones neonatos aparentemente sanos. Los genotipos PCV2a y PCV2b fueron detectados en la infección in utero. Conclusiones: las cerdas de reemplazo pueden estar infectadas con el PCV2 antes de ingresar a las explotaciones de cría o pueden infectarse por transmisión horizontal durante la cuarentena y gestación. La exposición e infección viral de las cerdas durante la gestación puede resultar en infección subclínica de los lechones neonatos.

Antecedentes: o circovírus suíno tipo 2 (PCV2) está associado a casos de falha reprodutiva em granjas recém-assentadas e fazendas de criação de marrãs e afeta principalmente a porcas nulíparas. Clinicamente observam-se aumento dos fetos abortados no segundo e terceiro estágios da gravidez, fetos mumificados, natimortos e nascimento de leitões inviáveis. Objetivo: o principal objetivo deste estudo foi avaliar a dinâmica da infeção pelo circovirus porcino tipo 2 e os títulos de anticorpos neutralizantes em porcas nulíparas com infecção subclínica e o efeito em sua leitegada. Métodos: este estudo foi realizado com 40 porcas em quatro granjas selecionadas aleatoriamente. Foram coletados de cada animal amostras de sangue, esfregaços nasais e vaginais ao entrar na fazenda, durante a quarentena, no parto e um dia pós-parto. Também foram coletadas amostras de colostro no parto e um dia pós-parto. Amostras de sangue, esfregaços nasal e tecido foram tomadas de quatro leitões antes de consumir colostro. As amostras foram analisadas pelo PCR Sybr Green para detectar e quantificar o PCV2. A detecção de anticorpos contra o vírus do PCV2 em soro foi realizada pelo teste de soroneutralização e todas as amostras foram analisadas através da técnica de SYBR Green PCR para a detecção do ADN viral. Resultados: a detecção de um nível elevado de viremia e a demonstração da excreção viral em esfregaços nasais e vaginais nas fêmeas permitiram demonstrar a introdução de porcas nulíparas aparentemente saudávels. Igualmente, o soro e as secreções vaginais e nasais foram positivos por PCR SYBR Green em tempo real na maioria das porcas no parto. Observou-se excreção viral em secreções vaginais e nasais e colostro na presença de anticorpos neutralizantes. A infecção das porcas manifestou-se no desenvolvimento de anticorpos neutralizantes e detecção de infecção fetal em leitões recém-nascidos aparentemente saudáveis , confirmando a transmissão vertical do PCV2. Os genótipos PCV2a e PCV2b foram detectados na infecção in utero. Conclusões: as porcas nulíparas podem estar infectadas com PCV2 antes de entrar nas granjas de criação e pode ser infectadas por transmissão horizontal durante a quarentena e gravidez. Exposição e infecção viral durante a gestação pode resultar em infecção subclínica de leitões recém-nascidos.

Comportamiento del virus de la bronquitis infecciosa aviar en aves con sintomatología respiratoria provenientes de granjas de producción del Departamento de Cundinamarca / Avian Infectious Bronchitis virus behavior in birds from commercial farms with a history of respiratory disease, located in the department of Cundinamarca

Objetivo. Evaluar la dinámica serológica contra el virus de bronquitis infecciosa aviar y su relación con la presentación y/o antecedentes de signos clínicos y hallazgos patológicos, bajo condiciones de campo. Materiales y métodos. Se realizó un muestreo al azar en dos fases, en pollo de engorde y reproductoras de granjas del Departamento de Cundinamarca. En la primera fase se tomó muestra de sangre a un total de 224 aves, distribuidas en 7 granjas. En la segunda fase, realizada 20 días posteriores al primer muestreo, se tomó una segunda muestra al mismo número de aves empleadas inicialmente. Las muestras de los sueros obtenidos se emplearon para la realización del inmunoensayo ligado a enzima (ELISA), diseñado para detectar anticuerpos frente al virus de la bronquitis infecciosa aviar en suero sanguíneo. Resultados. Se obtuvo que del total de las granjas analizadas el 85.72% mostró reactividad serológica al virus de la Bronquitis Infecciosa aviar (VBIA), con correlación ante la presencia de los signos clínicos o antecedentes respiratorios en granja.

Objective. Evaluate the serological dynamics against avian infectious bronchitis virus and its relationship with the presentation and / or a history of clinical signs and pathological findings, under field conditions. Materials and methods. A random sampling was conducted in two phases, in broiler and breeder farms located in the Department of Cundinamarca. In the first phase blood sample were taken from a total of 224 birds, distributed over the 7 farms. In the second phase, carried out 20 days after the first, a second sample was collected from the same number of birds used in the first phase. The serum samples were used to carry out the enzyme linked immunoassay (ELISA) intended to detect antibodies against avian infectious bronchitis virus in blood serum. Results. As a result it was found that from the total farms analyzed the 85.72% showed serologic reactivity against Avian Infectious Bronchitis Virus (AIBV) that correlated to the presence of clinical signs or previous history of respiratory disease in the farm.

Detection and molecular characterization of porcine circovirus type 2 from piglets with porcine circovirus associated diseases in Colombia.

BACKGROUND: The porcine circovirus-associated disease (PCVAD) has been known since 1991 in Canada, but the first outbreak of PCVAD in Colombia was reported in 2007. In order to understand the molecular epidemiology of the disease and to establish the origin of the virus in the country, the study presented here intended to evaluate the presence of PCV2-associated systemic infection in piglets from different geographical regions over a period of 9-years (2002-2010). The analysis included samples collected before, during and after outbreaks of PCVAD in pigs from Colombia. The PCV2 ORF2 from the positive samples was sequenced and used to determine the genotypes of the strains and to study the dynamic of these genotypes throughout the time. RESULTS: PCV2 DNA was detected in cases related to PCV2-associated systemic infection as well as in healthy pigs with a presumable persistent infection. The analysis of the ORF2 nucleotide full length sequence of twenty-three strains allowed to divide them into two groups: PCV2a and PCV2b. At the amino acid level the main variations in the sequence of the capsid protein were found in regions located within the immunoreactive areas. CONCLUSIONS: The results of this study demonstrated for the first time, that the two subgroups: PCV2a and PCV2b have been circulating in swine from Colombia. In addition, the study showed that genotype PCV2b is present in Colombian pigs suffering from both clinical and presumable persistent infection and that the PCV2b genotype was present in the Colombian pig population even before recognition of the disease in the country and it became predominant through time.

Association of swine influenza H1N1 pandemic virus (SIV-H1N1p) with porcine respiratory disease complex in sows from commercial pig farms in Colombia.

Porcine respiratory disease complex (PRDC) is a serious health problem that mainly affects growing and finishing pigs. PRDC is caused by a combination of viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Mycoplasma hyopneumoniae (Myh), Actinobacillus pleuropneumoniae (APP), Pasteurella multocida and Porcine circovirus 2 (PCV2). To characterize the specific role of swine influenza virus in PRDC presentation in Colombia, 11 farms from three major production regions in Colombia were examined in this study. Nasal swabs, bronchial lavage and lung tissue samples were obtained from animals displaying symptoms compatible with SIV. Isolation of SIV was performed in 9-day embryonated chicken eggs or Madin-Darby Canine Kidney (MDCK) cells. Positive isolates, identified via the hemagglutination inhibition test, were further analyzed using PCR. Overall, 7 of the 11 farms were positive for SIV. Notably, sequencing of the gene encoding the hemagglutinin (HA) protein led to grouping of strains into circulating viruses identified during the human outbreak of 2009, classified as pandemic H1N1-2009. Serum samples from 198 gilts and multiparous sows between 2008 and 2009 were obtained to determine antibody presence of APP, Myh, PCV2 and PRRSV in both SIV-H1N1p-negative and -positive farms, but higher levels were recorded for SIV-H1N1p-positive farms. Odds ratio (OR) and P values revealed statistically significant differences (p<0.05) in PRDC presentation in gilts and multiparous sows of farms positive for SIV-H1N1p. Our findings indicate that positive farms have increased risk of PRDC presentation, in particular, PCV2, APP and Myh.

Diferenciación de cepas de Mycoplasma gallisepticum a partir de RFLP / Differentiation of Mycoplasma gallisepticum Strains through RFLP

La micoplasmosis aviar es una enfermedad que afecta considerablemente al sector avícola, lo que se ve reflejado en la disminución de parámetros productivos en aves de engorde ponedoras de huevo fértil y ponedoras de huevo comercial. Los costos de su presentación son tan altos que es imposible la supervivencia de la avicultura industrial sin pensar en el control efectivo o en su erradicación. Existe un gran interés en la tipificación de cepas de M. gallisepticum(Mg) tanto vacunales como de campo, aspecto clave para el manejo de la enfermedad, pero aún no existe un método definitivo de caracterización de cepas de Mg. En la actualidad se están estudiando genes relacionados con proteínas de superficie —gapA y mgc2, lipoproteína (lp)— que permitan identificar y caracterizar genómicamente al Mg. En el presente estudio se amplificaron regiones del gen lp a partir de las cepas F y Ts-11 del Mg por la técnica de reacción en cadena de la polimeras a (PCR) que dio un amplicón de 455 pb para cada una de las cepas; a cada uno de las aplicaciones se les hizo la prueba de polimorfismo de longitud de los fragmentos de restricción (RFLP) con la enzima Taq I, lo que permitió diferenciar cepas vacunales de cepas de campo obtenidas a partir de muestras de hisopos traqueales tomados en granjas de explotación comercial. Se demostró que el PCR-RFLP es un método adecuado de diagnóstico de la micoplasmosis en nuestro medio...

Avian mycoplasmosis is a disease that considerably affects the poultry sector, which is reflectedin the decrease of the production parameters in fertile and commercial egg layingbroilers. Presentation costs are so high that it is impossible for the poultry industry to survivewithout thinking of its effective control or eradication. There is great interest in the type ofM. gallisepticum (Mg) strains, both vaccine and field, which are key aspects to handle thedisease, but there is still no definitive method for Mg strain characterization. Genes relatedto surface proteins —gapA and mgc2,lipoprotein (lp)— that make it possible to identify andcharacterize the Mg genomically are currently being studied. In this study, regions of the lpgene were amplified from strains F and Ts-11 of Mg through the polymerase chain reaction(PCR) technique, which gave an amplicon of 455 bp for each of the strains; each of amplicons was applied the restriction fragment length polymorphism (RFLP) test with theTaq I enzyme, which made it possible to differentiate vaccine strains from field strains obtainedfrom tracheal swab samples taken at commercial farms. It was demonstrated that PCRRFLPis an appropriate method of diagnosis of mycoplasmosis in our environment...

A micoplasmose aviar é uma doença que afeta consideravelmente o setor avícola, isso se refletena diminuição de parâmetros produtivos em aves de engorde poedeiras de ovo fértil epoedeiras de ovo comercial. Os custos de sua apresentação são tão altos que é impossível asobrevivência da avicultura industrial sem pensar no controle efetivo ou em sua erradicação.Existe um grande interesse na tipificação de cepas de M. gallisepticum (Mg) tanto vacinaiscomo de campo, aspecto chave para o manejo da doença, mas ainda não existe um métododefinitivo de caracterização de cepas de Mg. Na atualidade estão sendo estudados genes relacionadoscom proteínas de superfície —gapA e mgc2, lipoproteína (lp)— que permitamidentificar e caracterizar genômicamente ao Mg. No presente estudo se amplificaram regiõesdo gen lp a partir das cepas F e Ts-11 do Mg pela técnica de reação em cadeia da polimerase(PCR) que deu um amplicon de 455 pb para cada uma das cepas; a cada um dos ampliconsfoi feito o teste de polimorfismo de longitude dos fragmentos de restrição (RFLP) com aenzima Taq I, o que permitiu diferenciar cepas vacunais de cepas de campo obtidas a partirde mostras de mostras traqueais tomadas em granjas de exploração comercial. Demonstrou-seque o PCR-RFLP é um método adequado de diagnóstico da micoplasmose em nosso meio...

Adaptation of a mallard H5N2 low pathogenicity influenza virus in chickens with prior history of infection with infectious bursal disease virus.

The influenza A/Mallard/Pennsylvania/10218/1984 (H5N2) virus is unable to replicate in 3-wk-old immunocompetent specific-pathogen-free chickens when a dose of 5 x 10(6) 50% egg infectious dose/ml is used. In contrast, this mallard virus shows limited replication in 3-wk-old chickens that had been previously infected at 2 days of age with, and recovered from, the immunosuppressive agent infectious bursal disease virus (IBDV; herein referred to as IBDV chickens). This limited replication in IBDV chickens allowed for the serial passage of the mallard influenza virus in chickens. After 22 passages (P22) in IBDV chickens, the resulting chicken-adapted influenza virus replicated in both immunocompetent and IBDV chickens more efficiently than the mallard influenza virus. Analysis of the outcomes of infection and the lesions caused by the two viruses at the microscopic level in a time-point study showed that the P22 virus is more virulent than the parental mallard virus in both immunocompetent and IBDV chickens. Our studies provide evidence that a previous history of IBDV infection in chickens may render them more susceptible to avian influenza virus (AIV) infections, allowing for the potential introduction of AIVs in an otherwise resistant population.

Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection: Evidence of competitive advantage of pandemic H1N1 influenza versus seasonal influenza.

On June 11, 2009 the World Health Organization (WHO) declared a new H1N1 influenza pandemic. This pandemic strain is as transmissible as seasonal H1N1 and H3N2 influenza A viruses. Major concerns facing this pandemic are whether the new virus will replace, co-circulate and/or reassort with seasonal H1N1 and/or H3N2 human strains. Using the ferret model, we investigated which of these three possibilities were most likely favored. Our studies showed that the current pandemic virus is more transmissible than, and has a biological advantage over, prototypical seasonal H1 or H3 strains.

Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential.

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

Multivalent HA DNA vaccination protects against highly pathogenic H5N1 avian influenza infection in chickens and mice.

BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 microg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.