Genotyping of news variants of the avian infectious bronchitis virus from Tolima department, Colombia / Genotipificación de variantes del virus de bronquitis infecciosa aviar en el departamento del Tolima, Colombia

ABSTRACT Objective. The aim of this study was identify the different genotypes of infectious bronchitis virus (IVB) present in commercial poultry farms from different localities of the Tolima Department, Colombia. Materials and methods. 105 samples of tracheal swabs of poultry of 21 farms were collected. Poultry had been vaccinated against IVB. An screen to identify positive samples and posteriorly the sequencing of the partial region of the S1 subunit and phylogenetic analysis of the isolates with the reference strains, including the vaccine currently used in the country was performed. Results . Poultry all farms had respiratory signs, but only four farms was confirmed the disease. Positive samples of the IBV (HT6, HT9, HT10 and HT11) were pathogenic for embryos 9-days-old. The HT6 sample was grouped in the same cluster that the Massachusetts strains. The HT9 and HT11 samples showed 99% similarity and were grouped genetically distant from the reference strains and other isolated. The HT10 sample showed low similarities with the isolates and reference strains, grouping alone in another cluster. Conclusions . New genotypes are circulating in the Tolima Department, where there is a risk of genetic recombination. Is believed that vaccines used were not providing cross-protection against the new genotypes.

RESUMEN Objetivo. El objetivo de este estudio fue identificar los diferentes genotipos del virus de bronquitis infecciosa (VBI) presente en granjas avícolas comerciales de diferentes localidades del departamento de Tolima, Colombia. Materiales y métodos . Se recolectaron 105 muestras de hisopados traqueales provenientes de aves de 21 granjas. Las aves habían sido vacunadas contra el VBI. Se realizó un "screen" para identificar muestras positivas y posteriormente la secuenciación de la región parcial de la subunidad S1 y el análisis filogenético de los aislamientos con las cepas de referencia, incluidas las vacunas utilizadas actualmente en el país. Resultados. Aves de todas las granjas tenían signos respiratorios, pero sólo cuatro granjas confirmaron la enfermedad. Las muestras positivas de VBI (HT6, HT9, HT10 y HT11) fueron patogénicas para embriones de 9 días de edad. La muestra HT6 se agrupó en el mismo clúster que las cepas de la vacuna Massachusetts. Las muestras HT9 y HT11 mostraron 99% de similitud y agrupan genéticamente distantes de las cepas de referencia y de los aislados. El HT10 mostró baja similitud con aislamientos y cepas de referencia, agrupándose separadamente en otro clúster. Conclusiones. Nuevos genotipos circulan en el departamento del Tolima, donde hay un riesgo de recombinación genética. Se estima que las vacunas utilizadas no están ofreciendo una protección cruzada contra los nuevos genotipos.

Supplementation with humic substances affects the innate immunity in layer hens in posfasting phase / El suplemento de sustancias húmicas afecta la inmunidad innata en gallinas ponedoras en fase posmuda

Objective. Asses the effect of supplementation with Humic substances (HS) over some innate immunity parameters (serum bactericidal activity, phagocytosis, bacterial agglutination, respiratory burst and lisozyme activity) in phase after fasting of layer hens. Materials and methods. 120 posfasting phase Hy Line Brown layer hens were taken which were distributed into four groups: The first and the second were supplemented with 0.1 and 0.2% of HS, respectively. The third group was supplemented with 0.25 mg/kg on levamisole hydrochloride and fourth group have no supplementation; during sixty days period. Blood samples were collected on 8th, 30th and 60th of experiment day. Results. The phagocytic index and respiratory burst increased significantly at day 30th in HS supplemented groups. Alike, serum bactericidal activity and lisozyme activity improved on 8 th day, nevertheless, changes were no evident latter. The bacterial agglutination was high in supplemented groups evaluated at everyone times. Conclusions. Results showed that HS behave as immunostimulant in the early phase after fasting layer hens.

Objetivos. Evaluar el efecto de las sustancias húmicas (SH) sobre algunos parámetros de la inmunidad innata (actividad bactericida del suero, fagocitosis, aglutinación bacteriana, explosión respiratoria y actividad de la lisozima) en la fase posmuda de gallinas ponedoras. Materiales y métodos. Se utilizaron 120 gallinas ponedoras Hy Line Brown en la fase de posmuda, las cuales fueron divididas en cuatro grupos: Los dos primeros fueron suplementados con 0.1 y 0.2% de SH respectivamente, el tercer grupo fue suplementado con 0.25 mg/kg de Clorhidrato de levamisol y el cuarto grupo control sin suplemento; durante un período de 60 días. Las muestras sanguíneas se tomaron los días 8, 30 y 60 del experimento. Resultados. El Índice fagocítico y la explosión respiratoria se incrementaron significativamente a partir del día 30 de suplementación con SH. De la misma manera, la actividad bactericida del suero y la actividad de la lisozima aumentaron al día 8; no obstante no se evidenciaron cambios posteriores. La aglutinación bacteriana fue significativamente mayor en los grupos suplementados en todos los tiempos evaluados. Conclusiones. Los resultados demuestran que las SH se comportan como agentes inmunoestimulantes en la fase temprana de la posmuda en gallinas ponedoras Hy Line Brown.

Hipertensión y diarrea: un solo diagnóstico / Hypertension and diarrhea: a single diagnosis

Una mujer de 53 años fue remitida a nuestra consulta para un estudio de hipertensión. Se detectó elevación de catecolaminas, metanefrinas y vanilmandélico en orina. En tomografía axial computarizada abdominal se demostró un nódulo suprarrenal de 3,3 cm, compatible todo ello con feocromocitoma. Cuatro meses después la paciente consulta por un cuadro de diarrea acuosa y pérdida de peso. Tras la realización de adrenalectomía laparoscópica remitió el cuadro de diarrea. Ante la sospecha de vipoma (tumor secretor de péptido intestinal vasoactivo [VIP]) se procedió a la realización de VIP en la pieza quirúrgica mediante técnicas inmunohistoquímicas; el resultado fue positivo. El diagnóstico final fue de feocromocitoma secretor de VIP

A 53 year-old woman referred to our consultation for study of hypertension. Catecholamines, metanephrines and vanilmandelic acid in urine was detected. Abdominal CT scan demonstrated a 3.3 cm adrenal node, all with pheochromocytoma. Months after, the patient consulted for a picture of watery diarrhea and weight loss. After performing laparoscopic adrenalectomy, the diarrhea picture remitted. Due to the suspicion of vipoma (VIP secreting tumor), vasoactive intestinal peptide (VIP) was visualized in surgical pieces using immunohistochemical techniques, this being positive. The final diagnosis was VIP secreting pheochromocytoma

[New mood stabilizers and reproductive cycle]. / Nuevos eutimizantes y ciclo reproductivo.

INTRODUCTION: The use of new mood stabilizers in the treatment of bipolar disorder has supposed a revolution, especially due to its more favorable profile in many aspects. Nevertheless, therapeutic decisions on treatment during pregnancy and the breastfeeding period are still being debated. Since these new anticonvulsants appeared in the decade of 1990, less naturalistic experience in its use exists during these periods than with other older anticonvulsants. METHODS: A Medline and Embase search was conducted from 1970 to 2003 to review the articles published on the use of the new mood stabilizers during pregnancy and breastfeeding, and its effects on contraception. Neurology and psychiatry text chapters and abstracts of the Annual Meeting of the American Psychiatric Association (years 2001-2003) were also reviewed. RESULTS: Although some recent articles suggest that new mood stabilizers could have a smaller risk of congenital defects, and therefore could be used with smaller risks in women in fertile age, most of articles reviewed indicate that there is not enough knowledge on the safety of the new mood stabilizers in pregnancy and breastfeeding. CONCLUSIONS: The risks and benefits of continuing with the new mood stabilizers during pregnancy and breastfeeding must be weighed carefully, and the severity of the disease and the previous answer to treatment should be taken into account.

Nuevos eutimizantes y ciclo reproductive / New mood stabilizers and reproductive cycle

Introducción. La utilización de los nuevos eutimizantes en el tratamiento del trastorno bipolar ha supuesto una revolución, especialmente debido a su perfil más favorable en muchos aspectos; sin embargo, las decisiones terapéuticas acerca del tratamiento durante la gestación y el período de lactancia siguen siendo controvertidas. Dado que estos nuevos anticonvulsivos aparecieron en la década de 1990, existe menos experiencia naturalística en su uso durante la gestación que con otros anticonvulsivos más antiguos. Métodos. Se efectuó una búsqueda bibliográfica en Medline y Embase desde 1970 hasta 2003, para localizar los artículos publicados en torno a la utilización de los nuevos eutimizantes en la gestación y lactancia y a sus efectos sobre la anticoncepción. También se revisaron capítulos de textos de neurología y psiquiatría y los resúmenes de los años 2001-2003 de la Reunión Anual de la Asociación Americana de Psiquiatría. Resultados. Aunque algunos estudios de publicación reciente sugieren que los nuevos estabilizantes podrían poseer un menor riesgo de defectos congénitos y por tanto podrían ser usados con menores riesgos en mujeres en edad fértil, la mayoría de los artículos revisados coinciden en señalar que no existen conocimientos suficientes acerca de la seguridad de los nuevos eutimizantes en la gestación y en la lactancia. Conclusiones. Los riesgos y beneficios de continuar con los nuevos estabilizantes durante los períodos e gestación y lactancia deben ser sopesados cuidadosamente, y debería tenerse en cuenta la gravedad de la enfermedad y la respuesta previa al tratamiento (AU)

Introduction. The use of new mood stabilizers in the treatment of bipolar disorder has supposed a revolution, especially due to its more favorable profile in many aspects. Nevertheless, therapeutic decisions on treatment during pregnancy and the breastfeeding period are still being debated. Since these new anticonvulsants appeared in the decade of 1990, less naturalistic experience in its use exists during these periods than with other older anticonvulsants. Methods. A Medline an Embase search was conducted from 1970 and 2003 to review the articles published on the use of the new mood stabilizers during pregnancy and breastfeeding, and its effects on contraception. Neurology and psychiatry text chapters and abstracts of the Annual Meeting of the American Psychiatric Association (years 2001-2003) were also reviewed. Results. Although some recent articles suggest that new mood stabilizers could have a smaller risk of congenital defects, and therefore could be used with smaller risks in there is not enough knowledge on the safety of the new mood stabilizers in pregnancy and breastfeeding. Conclusions. The risks and benefits of continuing with the new mood stabilizers during pregnancy and breastfeeding must be weighed carefully, and the severity of the disease and the previous answer to treatment should be taken into account (AU)

Enrichment of gene-coding sequences in maize by genome filtration.

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C0t selection were sequenced to generate approximately 200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

Cereal genes similar to Snf2 define a new subfamily that includes human and mouse genes.

Genes from the SNF2 family play important roles in transcriptional regulation, maintenance of chromosome integrity and DNA repair. This study describes the molecular cloning and characterization of cereal genes from this family. The predicted proteins exhibit a novel C-terminal domain that defines a new subfamily designated SNF2P that includes human and mouse proteins. Comparison between genomic and cDNA sequences showed that cereal Snf2P genes consisted of 17 exons, including one only 8 bp long. Two barley alleles differed by the presence of a 7.7-kb non-LTR retrotransposon in intron 6. An alternative annotation of the orthologous Arabidopsis gene would improve its similarity with the other members of the subfamily. Intron 2 was not spliced out in approximately half of the rice Snf2P mRNAs present in leaves, resulting in a premature stop codon. Transcripts from the barley and wheat Snf2P genes were found in apexes, leaves, sheaths, roots and spikes. The Snf2P genes exist as single copies on wheat chromosome arm 5A(m)L and in the colinear regions on barley chromosome arm 4HL and rice chromosome 3. High-density genetic mapping and RT-PCR suggest that Snf2P is not a candidate gene for the tightly linked vernalization gene Vrn2.

Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes.

Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3' untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.

Colinearity and its exceptions in orthologous adh regions of maize and sorghum.

Orthologous adh regions of the sorghum and maize genomes were sequenced and analyzed. Nine known or candidate genes, including adh1, were found in a 225-kilobase (kb) maize sequence. In a 78-kb space of sorghum, the nine homologues of the maize genes were identified in a colinear order, plus five additional genes. The major fraction of DNA in maize, occupying 166 kb (74%), is represented by 22 long terminal repeat (LTR) retrotransposons. About 6% of the sequence belongs to 33 miniature inverted-repeat transposable elements (MITEs), remnants of DNA transposons, 4 simple sequence repeats, and low-copy-number DNAs of unknown origin. In contrast, no LTR retroelements were detected in the orthologous sorghum region. The unconserved sorghum DNA is composed of 20 putative MITEs, transposon-like elements, 5 simple sequence repeats, and low-copy-number DNAs of unknown origin. No MITEs were discovered in the 166 kb of DNA occupied by the maize LTR retrotransposons. In both species, MITEs were found in the space between genes and inside introns, indicating specific insertion and/or retention for these elements. Two adjacent sorghum genes, including one gene missing in maize, had colinear homologues on Arabidopsis chromosome IV, suggesting two rearrangements in the sorghum and three in the maize genome in comparison to a four-gene region of Arabidopsis. Hence, multiple small rearrangements may be present even in largely colinear genomic regions. These studies revealed a much higher degree of diversity at a microstructural level than predicted by genetic mapping studies for closely related grass species, as well as for comparisons of monocots and dicots.

The paleontology of intergene retrotransposons of maize.

Retrotransposons, transposable elements related to animal retroviruses, are found in all eukaryotes investigated and make up the majority of many plant genomes. Their ubiquity points to their importance, especially in their contribution to the large-scale structure of complex genomes. The nature and frequency of retro-element appearance, activation and amplification are poorly understood in all higher eukaryotes. Here we employ a novel approach to determine the insertion dates for 17 of 23 retrotransposons found near the maize adh1 gene, and two others from unlinked sites in the maize genome, by comparison of long terminal repeat (LTR) divergences with the sequence divergence between adh1 in maize and sorghum. All retrotransposons examined have inserted within the last six million years, most in the last three million years. The structure of the adh1 region appears to be standard relative to the other gene-containing regions of the maize genome, thus suggesting that retrotransposon insertions have increased the size of the maize genome from approximately 1200 Mb to 2400 Mb in the last three million years. Furthermore, the results indicate an increased mutation rate in retrotransposons compared with genes.

Grass genomes.

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Sequence organization and conservation in sh2/a1-homologous regions of sorghum and rice.

Previously, we have demonstrated microcolinearity of gene composition and orientation in sh2/a1-homologous regions of the rice, sorghum, and maize genomes. However, the sh2 and a1 homologues are only about 20 kb apart in both rice and sorghum, while they are separated by about 140 kb in maize. In order to further define sequence organization and conservation in sh2/a1-homologous regions, we have completely sequenced a 42,446-bp segment of sorghum DNA. Four genes were identified: a homologue of sh2, two homologues of a1, and a putative transcriptional regulatory gene. A solo long terminal repeat of the retroelement Leriathan was detected between the two a1 homologues, and eight miniature inverted repeat transposable elements were found in this region. Comparison of the sorghum sequence with the sequence of the homologous segment from rice indicated that only the identified genes were evolutionarily conserved between these two species, which have evolved independently for over 50 million years. The introns of the a1 homologues have evolved faster than the introns of the sh2 homologue. The a1 tandem duplication appears to be an ancient event that may have preceded the ancestral divergence of maize, sorghum, and rice.

Demonstration of vertical transmission of Streptococcus suis in swine by genomic fingerprinting.

Isolates of Streptococcus suis serotype 5 collected from three sows and nine of their pigs at birth were analyzed by genomic DNA fingerprinting. The cleavage patterns of DNA from S. suis isolated from the sows matched the cleavage patterns of DNA from S. suis isolated from their respective pigs.

Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes.

Large regions of genomic colinearity have been demonstrated among grass species by recombinational mapping, but the degree of chromosomal conservation at the sub-centimorgan level has not been extensively investigated. We cloned the rice and sorghum genes homologous to the sh2 locus of maize on bacterial artificial chromosomes (BACs), and observed that a homologue of the maize a1 gene was also present on each of these BACs. In sorghum, we found a direct duplication of a1 homologues separated by about 10 kb. In maize, sh2 and a1 are approximately 140 kb apart and transcribed in the same direction, with sh2 upstream of a1. In rice and sorghum, this arrangement is fully conserved. However, the sh2 and a1 homologues are separated by about 19 kb in both rice and sorghum. We found low-copy-number and repetitive DNAs between the sh2 and a1 homologues of sorghum and rice. The sh2 and a1 homologues cross-hybridized, but the repetitive DNA and most low-copy-number sequences between these genes did not. These results indicate that maize, sorghum, and rice have conserved gene order and composition in the sh2-a1 region, but have acquired extensive qualitative and quantitative differences in the sequences between these genes.

Gene identification in a complex chromosomal continuum by local genomic cross-referencing.

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.

Nested retrotransposons in the intergenic regions of the maize genome.

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

The Hybaid Lecture. Microcollinearity and segmental duplication in the evolution of grass nuclear genomes.

Recent studies have shown that grass genomes have very similar gene compositions and regions of conserved gene order, as exemplified by collinear genetic maps of DNA markers. We have begun the detailed study of sequence organization in large (100-500 kb) segments of the nuclear genomes of maize, sorghum and rice. Our results indicate collinearity of genes in the regions homoeologous to the maize adh1 and sh2-a1 genes. Comparable genes were found to be physically closer to each other in grasses with small genomes (rice and sorghum) than they are in maize. In several instances, we have found evidence of tandem and 'distantly tandem' duplications of segments containing maize and sorghum genes. These duplications complicate characterizations of microcollinearity and could also interfere with some map-based approaches to gene isolation.

Matrix attachment regions and transcribed sequences within a long chromosomal continuum containing maize Adh1.

We provide evidence for the location of matrix attachment sites along a contiguous region of 280 kb on maize chromosome 1. We define nine potential loops that vary in length from 6 kb to > 75 kb. The distribution of the different classes of DNA within this continuum with respect to the predicted structural loops reveals an interesting correlation: the long stretches of mixed classes of highly repetitive DNAs are often segregated into topologically sequestered units, whereas low-copy-number DNAs (including the alcohol dehydrogenase1 [adh1] gene) are positioned in separate loops. Contrary to expectations, several classes of highly repeated elements with representatives in this region were found to be transcribed, and some of these exhibited tissue-specific patterns of expression.

Active maize genes are unmodified and flanked by diverse classes of modified, highly repetitive DNA.

We have characterized the copy number, organization, and genomic modification of DNA sequences within and flanking several maize genes. We found that highly repetitive DNA sequences were tightly linked to most of these genes. The highly repetitive sequences were not found within the coding regions but could be found within 6 kb either 3' or 5' to the structural genes. These highly repetitive regions were each composed of unique combinations of different short repetitive sequences. Highly repetitive DNA blocks were not interrupted by any detected single copy DNA. The 13 classes of highly repetitive DNA identified were found to vary little between diverse Zea isolates. The level of DNA methylation in and near these genes was determined by scoring the digestibility of 63 recognition/cleavage sites with restriction enzymes that were sensitive to 5-methylation of cytosines in the sequences 5'-CG-3' and 5'-CNG-3'. All but four of these sites were digestible in chromosomal DNA. The four undigested sites were localized to extragenic DNA within or near highly repetitive DNA, while the other 59 sites were in low copy number DNAs. Pulsed field gel analysis indicated that the majority of cytosine modified tracts range from 20 to 200 kb in size. Single copy sequences hybridized to the unmodified domains, while highly repetitive sequences hybridized to the modified regions. Middle repetitive sequences were found in both domains.