Development of a Coccidiosis Disease Challenge Model Using a Commercially Available Live Oocyst Vaccine.

With growing cross-disciplinary collaboration among researchers, it is increasingly important to record detailed methodology to prevent the repetition of preliminary experiments. The purpose of this paper is to explain the development of a coccidiosis challenge model for the investigation of dietary interventions to coccidiosis in broiler chickens. The objectives are to select a dose of mixed species coccidial vaccine and evaluate the suitability (ability to produce a consistent, marked change) of selected response variables important to nutritional studies at different times postinfection (PI). Coccivac-B and Coccivac-B52 (Merck Animal Health) were evaluated as the source of coccidia in three trials. Trials 1 and 2 were randomized complete block designs with four doses (0, 10, 20, or 30 times (×) label dose) of Coccivac-B administered to 12 replicate cages of six birds by repeater pipette (Trial 1) or gavaging needle (Trial 2). Trial 3 used a completely randomized design with 0× or 30× label dose of Coccivac-B52 administered by gavaging needle to six replicate cages of six birds. Birds were gavaged at 15 days of age, and response criteria were evaluated 7 days PI in all trials and again at 10 days PI in Trials 1 and 2. All means are reported in order of increasing coccidia dose with significance accepted at P ≤ 0.05. Broiler performance was not affected by coccidia in Trials 1 or 3 but grew poorer with increasing dose from 0 to 7 days PI in Trial 2 (body weight gain, 465, 421, 388, 365 g; feed to gain, 1.37, 1.47, 1.52, 1.58). As coccidia dose increased, nitrogen corrected apparent metabolizable energy decreased (Trial 1, 3387, 3318, 3267, 3170 kcal kg-1; Trial 2, 3358, 2535, 2422, 2309 kcal kg-1; Trial 3, not measured), while relative weight, length, and content for intestinal sections increased (Trials 1through 3). Gross lesion (duodenum, jejunum/ileum, ceca) and oocyst count scores (jejunum/ileum, ceca) increased with dose; however, gross scoring often suggested infection in unchallenged birds, a finding unsupported by oocyst count scores. At 7 days PI there was no correlation between midgut gross lesion score and midgut oocyst count score (r = 0.06, P = 0.705), but cecal scores were weakly correlated (r = 0.55, P < 0.001). Administering coccidia via repeater pipette (Trial 1) resulted in respiratory distress in some birds, while use of the gavaging needle (Trials 2 and 3) successfully induced intestinal damage in chickens without resulting in coccidia related mortality. Thirty times the label dose at 7 days PI resulted in the greatest number of response variables that produced a consistent, marked change. Therefore, consideration should be given to these conditions when designing future coccidiosis challenge models using vaccines as a source of coccidia.

Artículo regular­Desarrollo de un modelo de desafío para coccidiosis utilizando una vacuna de ooquistes vivos disponible comercialmente. Con la creciente colaboración interdisciplinaria entre investigadores, es cada vez más importante registrar la metodología detallada para evitar la repetición de experimentos preliminares. El propósito de este artículo es explicar el desarrollo de un modelo de desafío de coccidiosis para la investigación de intervenciones dietéticas para coccidiosis en pollos de engorde. Los objetivos son seleccionar una dosis de vacuna coccidial de especies mixtas y evaluar la idoneidad (capacidad de producir un cambio marcado y consistente) de las variables de respuesta seleccionadas que son importantes para los estudios nutricionales en diferentes momentos posteriores a la infección (PI). Las vacunas Coccivac-B o Coccivac B-52 (Merck Animal Health) se evaluaron como fuente de coccidias en tres ensayos. Los ensayos 1 y 2 fueron diseños de bloques completamente aleatorios con cuatro dosis (0, 10, 20 o 30 veces (×) la dosis indicada en la etiqueta) de Coccivac-B administradas a 12 jaulas repetidas de seis aves mediante una pipeta repetidora (ensayo 1) o por sonda oral. (Prueba 2). El ensayo 3 utilizó un diseño completamente aleatorio con una dosis de etiqueta de 0 × o 30 × de Coccivac-B52 administrada con una sonda oral en seis jaulas repetidas de seis aves. Las aves fueron inoculadas por sonda a los 15 días de edad y los criterios de respuesta se evaluaron a los 7 días postinoculación en todos los ensayos y nuevamente a los 10 días postinoculación en los ensayos 1 y 2. Todos los promedios se reportan en orden de dosis crecientes de coccidias con significancia aceptada en P ≤ 0.05. El rendimiento de los pollos de engorde no se vio afectado por las coccidias en los Ensayos 1 o 3, pero empeoró al aumentar la dosis de los cero a 7 días después de la inoculación en el Ensayo 2 (aumento de peso corporal, 465, 421, 388, 365 g; alimento para ganar, 1.37, 1.47, 1.52, 1.58). A medida que aumentaba la dosis de coccidia, la energía metabolizable de nitrógeno aparente y corregida disminuyó (Prueba 1, 3387, 3318, 3267, 3170 kcal kg-1; Prueba 2, 3358, 2535, 2422, 2309 kcal kg-1; Prueba 3, no medida), mientras que el peso relativo, la longitud y el contenido de las secciones intestinales aumentaron (ensayos 1 a 3). La lesión macroscópica (duodeno, yeyuno/íleon, ciego) y las puntuaciones del recuento de oocistos (yeyuno/íleon, ciego) aumentaron con la dosis; sin embargo, la puntuación bruta a menudo sugirió infección en aves no desafiadas, un hallazgo que no está respaldado por las puntuaciones del recuento de ooquistes. A los 7 días después de la infección no hubo correlación entre la puntuación de la lesión macroscópica del intestino medio y la puntuación del recuento de oocistos del intestino medio (r= 0,06, P= 0,705), pero las puntuaciones cecales se correlacionaron débilmente (r = 0.55, P <0.001). La administración de coccidias a través de una pipeta repetidora (Ensayo 1) provocó dificultad respiratoria en algunas aves, mientras que el uso de la sonda oral (Ensayos 2 y 3) indujo con éxito el daño intestinal en los pollos sin dar como resultado mortalidad relacionada con los coccidias. Treinta veces la dosis de la etiqueta a los 7 días después de la infección resultó en el mayor número de variables de respuesta que produjeron un cambio marcado y consistente. Por lo tanto, deben tenerse en cuenta estas condiciones al diseñar futuros modelos de exposición a la coccidiosis que utilicen vacunas como fuente de coccidias.

Maintenance of ancestral sex chromosomes in Palearctic tree frogs: direct evidence from Hyla orientalis.

Contrasting with the situation found in birds and mammals, sex chromosomes are generally homomorphic in poikilothermic vertebrates. This homomorphy was recently shown to result from occasional X-Y recombinations (not from turnovers) in several European species of tree frogs (Hyla arborea, H. intermedia and H. molleri). Because of recombination, however, alleles at sex-linked loci were rarely diagnostic at the population level; support for sex linkage had to rely on multilocus associations, combined with occasional sex differences in allelic frequencies. Here, we use direct evidence, obtained from anatomical and histological analyses of offspring with known pedigrees, to show that the Eastern tree frog (H. orientalis) shares the same pair of sex chromosomes, with identical patterns of male heterogamety and complete absence of X-Y recombination in males. Conservation of an ancestral pair of sex chromosomes, regularly rejuvenated via occasional X-Y recombination, seems thus a widespread pattern among Hyla species. Sibship analyses also identified discrepancies between genotypic and phenotypic sex among offspring, associated with abnormal gonadal development, suggesting a role for sexually antagonistic genes on the sex chromosomes.

Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup).

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X-Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex-linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X-Y recombination in males. Phylogenetic analyses of sex-linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X-Y homomorphy and fine-scale sequence similarity in these species do not stem from recent sex-chromosome turnovers, but from occasional X-Y recombination.

Distinct signal/response mechanisms regulate pax1 and QmyoD activation in sclerotomal and myotomal lineages of quail somites.

Pax1 and QmyoD are early sclerotome and myotome-specific genes that are activated in epithelial somites of quail embryos in response to axial notochord/neural tube signals. In situ hybridization experiments reveal that the developmental kinetics of activation of pax1 and QmyoD differ greatly, suggesting that myotome and sclerotome specification are controlled by distinct developmental mechanisms. pax1 activation always occurs in somite IV throughout development, indicating that pax1 regulation is tightly coordinated with early steps in somite maturation. In contrast, QmyoD is delayed and does not occur until embryos have 12-14 somites. At this time, QmyoD is the first of the myogenic regulatory factor (MRF) genes to be activated in preexisting somites in a rapid, anterior to posterior progression until the 22 somite stage, after which time QmyoD is activated in somite I immediately following somite formation. Experiments involving transplantation of newly formed somites to ectopic sites along the anterior to posterior embryonic axis were performed to distinguish the contributions of axial signals and somite response pathways to the developmental regulation of pax1 and QmyoD. These studies show that pax1 activation is regulated by somite formation and maturation, not by the availability of axial signals, which are expressed prior to somite formation. In contrast, the delayed activation of QmyoD is controlled by developmental regulation of the production of axial signals as well as by the competence of somites to respond to these signals. These somite transplantation studies, therefore, provide a basis for understanding the different developmental kinetics of activation of pax1 and QmyoD during sclerotome and myotome specification, and suggest specific molecular models for the developmental regulation of myotome and sclerotome formation in somites through distinct signal/response pathways.

Identification and analysis of fipA, a Fusobacterium nucleatum immunosuppressive factor gene.

We have previously demonstrated that sonic extracts of Fusobacterium nucleatum FDC 364 were capable of inhibiting human T-cell responses to mitogens and antigens. The purified F. nucleatum immunosuppressive protein (FIP) is composed of two subunits of 44 and 48 kDa. Furthermore, FIP inhibits T-cell activation by arresting cells in the middle of the G(1) phase of the cell cycle; the data available to date suggest that FIP impairs the expression of the proliferating-cell nuclear antigen. To initiate delineation of FIP structure-function relationships, molecular cloning of the FIP gene was carried out. A DNA library of F. nucleatum FDC 364 was constructed by partial digestion of genomic DNA with Sau3A and screened for the production of FIP with polyclonal antibody. Twelve immunoreactive clones were identified. One of these clones contained a 3.1-kbp insert and was chosen for further study. Cell lysates were found to contain an immunoreactive band that comigrated with the 44-kDa subcomponent of the native FIP. Sequencing of the 3.1-kpb insert revealed the presence of three open reading frames (ORFs). One ORF extends from nucleotides 415 to 1620, encodes 402 amino acids, and is preceded by a ribosome-binding site. Deletion analysis and antibody elution analysis showed that this ORF encodes the 44-kDa subunit (FipA) of native FIP. A second ORF is situated upstream of fipA. However, Northern (RNA) analysis suggested that fipA is not transcribed as part of an operon but transcribed from its own promotor. Finally, the partially purified recombinant FipA protein was capable of impairing T-cell activation in a manner consistent with the native protein. These results indicate that the two components that form the native protein are most probably distinct gene products and suggest that the 44-kDa FipA polypeptide is sufficient to mediate the immunosuppressive activities of the native protein complex.