Background: Marek's disease (MD) is a lymphoproliferative disease in chickens caused by Marek's disease virus (MDV) and characterized by T cell lymphoma and infiltration of lymphoid cells into various organs such as liver, spleen, peripheral nerves and muscle. Resistance to MD and disease risk have long been thought to be influenced both by genetic and environmental factors, the combination of which contributes to the observed outcome in an individual. We hypothesize that after MDV infection, genes related to MD-resistance or -susceptibility may exhibit different trends in transcriptional activity in chicken lines having a varying degree of resistance to MD.Results: In order to study the mechanisms of resistance and susceptibility to MD, we performed genome-wide temporal expression analysis in spleen tissues from MD-resistant line 6₃, susceptible line 7₂and recombinant congenic strain M (RCS-M) that has a phenotype intermediate between lines 6₃and 7₂after MDV infection. Three time points of the MDV life cycle in chicken were selected for study: 5 days post infection (dpi), 10dpi and 21dpi, representing the early cytolytic, latent and late cytolytic stages, respectively. We observed similar gene expression profiles at the three time points in line 6₃and RCS-M chickens that are both different from line 7₂. Pathway analysis using Ingenuity Pathway Analysis (IPA) showed that MDV can broadly influence the chickens irrespective of whether they are resistant or susceptible to MD. However, some pathways like cardiac arrhythmia and cardiovascular disease were found to be affected only in line 7₂; while some networks related to cell-mediated immune response and antigen presentation were enriched only in line 6₃and RCS-M. We identified 78 and 30 candidate genes associated with MD resistance, at 10 and 21dpi respectively, by considering genes having the same trend of expression change after MDV infection in lines 6₃and RCS-M. On the other hand, by considering genes with the same trend of expression change after MDV infection in lines 7₂and RCS-M, we identified 78 and 43 genes at 10 and 21dpi, respectively, which may be associated with MD-susceptibility.Conclusions: By testing temporal transcriptome changes using three representative chicken lines with different resistance to MD, we identified 108 candidate genes for MD-resistance and 121 candidate genes for MD-susceptibility over the three time points. Genes included in our resistance or susceptibility genes lists that are also involved in more than 5 biofunctions, such asCD8α,IL8,USP18, andCTLA4, are considered to be important genes involved in MD-resistance or -susceptibility. We were also able to identify several biofunctions related with immune response that we believe play an important role in MD-resistance.

Marek’s disease virus (MDV) is an oncovirus that induces lymphoid tumors in susceptible chickens, and may affect the epigenetic stability of the CD4 gene. The purpose of this study was to find the effect of MDV infection on DNA methylation status of the CD4 gene differed between MD-resistant (L63) and –susceptible (L72) chicken lines.

The objective was to evaluate lymphoid organ size in chickens from a series of 13 recombinant congenic strains (RCS) and their highly inbred parental lines (6(₃) and 7(₂)). The parental line 6(₃) was selected for resistance to tumors induced by Marek's disease virus and avian leukosis viruses, whereas line 7(₂) was selected for susceptibility to these tumors. Each RCS on the average contains a random one-eighth of genome from the donor line 7(₂). Previous studies have shown that lines 6(₃) and 7(₂) differ in the size of primary lymphoid organs; i.e., the bursa of Fabricius (BF) and the lobes of the thymus (T) are smaller in line 6(₃) than line 7(₂). In the current study, the relative size of the T, BF, and spleen was first examined in about 15 males from each of 13 RCS and the 2 parental lines at 60 to 69 d of age. The differences of relative BF, T, and spleen size among the RCS and the parental lines 6(₃) and 7(₂) differed significantly (P < 0.001). Males and females from 4 RCS and the 2 parental lines were evaluated a second time, and differences in the relative sizes in lymphoid organs among the RCS and parental lines were consistent. In 2 RCS, the size of the T and BF was comparatively large as in line 7(₂), leading to the conclusion that different allelic forms at 1 or more loci in these RCS regulate the size of both organs. In 2 other RCS, the BF was large compared with the T, suggesting that allelic forms at some loci in these RCS influence the BF independent of the T. The relative lymphoid organ size among the RCS appeared to cosegregate with the concentration of IgG in the plasma measured previously. The evaluation of genomic variability of these lines is underway, and the RCS are available for research on traits that differ between lines 6(₃) and 7(₂).

As a low pathogenic influenza virus, avian influenza virus subtype H9N2 (H9N2 AIV) often induces high morbidity in association with secondary bacterial infections in chickens or mammals. To explore this phenomenon, the relationship between intestinal microflora changes and bacterial translocations was studied post H9N2 AIV challenge and post AIV infection plus Ageratum-liquid treatment. Illumina sequencing, histological examination and Neongreen-tagged bacteria were used in this study to research the microbiota composition, intestinal barrier, and bacterial translocation in six weeks of BALB/c mice. H9N2 AIV infection caused intestinal dysbacteriosis and mucosal barrier damages. Notably, the villus length was significantly reduced (p < 0.01) at 12 dpi and the crypt depth was significantly increased (p < 0.01) at 5 dpi and 12 dpi with infection, resulting in the mucosal regular villus-length/crypt-depth (V/C) was significantly reduced (p < 0.01) at 5 dpi and 12 dpi. Moreover, degeneration and dissolution of the mucosal epithelial cells, loose of the connective tissue and partial glandular atrophy were found in infection group, indicating that intestinal barrier function was weakened. Eventually, intestinal microbiota (Staphylococcus, E. coli, etc.) overrun the intestinal barrier and migrated to liver and lung tissues of the mice at 5 and 12 dpi. Furthermore, the bacteria transferred in mesentery tissue sites from intestine at 36 h through tracking the Neongreen-tagged bacteria. Then the Neongreen-tagged bacteria were isolated from liver at 48 h post intragastrical administration. Simultaneously, Ageratum-liquid could inhibit the intestinal microbiota disorder post H9N2 AIV challenge via the respiratory tract. In addition, this study also illustrated that Ageratum-liquid could effectively prevent intestinal bacterial translocation post H9N2 AIV infection in mice. In this study, we report the discovery that H9N2 AIV infection could damage the ileal mucosal barrier and induce the disturbance of the intestinal flora in BALB/c mice resulting in translocation of intestinal bacteria. In addition, this study indicated that Ageratum-liquid can effectively prevent bacterial translocation following H9N2 infection. These findings are of important theoretical and practical significance in prevention and control of H9N2 AIV infection.

The cellular receptor of subgroup B avian leukosis virus (ALVB) is encoded by a gene at the tumour virus B (TVB) locus. TVB alleles encode specific receptors permitting infection by exogenous ALVB or avian leukosis virus subgroup D (ALVD) as well as endogenous avian leukosis virus subgroup E (ALVE), and thus susceptibility is dominant to resistance. Two single nucleotide polymorphisms at the TVB locus have been reported distinguishing three TVB alleles (TVB*S1, TVB*S3 and TVB*R). We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using the two single nucleotide polymorphisms to define three observed allelic haplotypes and to identify the six possible TVB genotypes consisting of the three haplotypes in defined laboratory strains of chickens. One additional potential allelic haplotype and four genotypes were also briefly discussed. Chickens from parents heterozygous for different TVB alleles were challenged with Rous sarcoma viruses of subgroup ALVB and ALVE to induce wing-web tumours. Tumour incidences were evaluated between chickens of the genotypes determined with this newly developed PCR-RFLP assay. Importantly, chickens typed with this assay as TVB*S3/*S3 were resistant to infection by ALVE only, and those TVB*R/*R were resistant to both ALVE and ALVB. Furthermore, a vast majority of chickens with the susceptible TVB*S1/– genotypes developed a tumour. This PCR-RFLP assay enables a relatively rapid assessment of all six anticipated TVB genotypes in experimental strains of chickens undergoing segregation for TVB*S1, TVB*S3, and TVB*R alleles. This non-infectious assay should be further evaluated for the capacity to select and breed commercial chickens for genetic resistance to infections by ALVB, ALVD and ALVE. Développement et validation d'un test PCR-RFLP pour évaluer les haplotypes TVB codant les récepteurs des sous groupes B et E des virus de la leucose aviaire chez les Leghorn blanche Le récepteur cellulaire du sous groupe B du virus de la leucose aviaire (ALVB) est encodé par un gène au niveau du locus tumeur virus B (TVB). Les allèles TVB encodent les récepteurs spécifiques permettant l'infection par les virus exogènes appartenant au sous groupe B, ou au sous groupe D (ALVD) ainsi que par les virus endogènes appartenant au sous groupe E (ALVE) et ainsi la sensibilité est dominante à résistante. Deux polymorphismes nucléotidiques simples (SNP) au niveau du locus TVB ont été rapportés distinguant trois allèles TVB (TVB*S1, TVB*S3 et TVB*R). Nous avons développé un test PCR-RFLP utilisant les deux SNPs pour définir trois haplotypes allèliques observés et pour identifier les six génotypes TVB possibles consistant en trois haplotypes définis chez les souches de poulet du laboratoire. Un haplotype allèlique complementaire et quatre génotypes ont également fait l'objet d'une courte discussion. Les poulets, issus de parents hétérozygotes pour différents allèles TVB, ont été éprouvés avec les virus du sarcome de Rous (RSVs) des sous groupes ALVB et ALVE pour induire des tumeurs à l'aile. Les incidences des tumeurs ont été évaluées chez les poulets de génotypes déterminés à l'aide du nouveau test développé PCR-RFPL. De façon importante, les poulets typés avec ce test comme étant TVB*S3/*S3 ont été résistants uniquement à l'infection par l'ALVE, et ceux typés TVB*R/*R ont été résistants aux deux virus ALVE et ALVB. De plus, une forte majorité de poulets avec les génotypes TVB*S1/- ont développé une tumeur. Ce test PCR-RFLP permet une évaluation relativement rapide des 6 génotypes TVB attendus chez les souches expérimentales de poulets en examinant la ségrégation des allèles TVB*S1, TVB*S3, et TVB*R. La capacité de ce test à sélectionner les croisements commerciaux de poulets pour leur résistance aux infections par des ALVB, ALVD, et ALVE doit être davantage évalué. Entwicklung und Validierung eines PCR-RFLP-Tests zur Untersuchung von TVB-Haplotypen mit kodierenden Rezeptoren für die aviären Leukoseviren der Subgruppen B und E bei weißen Leghorn-Hühnern Der zelluläre Rezeptor des aviären Leukosevirus Subgruppe B (ALVB) wird von einem Gen auf dem Tumorvirus B (TVB) kodiert. TVB-Allele kodieren spezifische Rezeptoren, die die Infektion mit exogenen ALVB oder ALV Subgruppe D (ALVD) sowie mit endogenenem ALV Subgruppe E (ALVE) zulassen, und deshalb ist der Empfänglichkeitsfaktor dominant über dem Resistenzfaktor. Der beschriebene Polymorphismus bei zwei einzelnen Nukleotiden (SNP) am TVB-Lokus ermöglichte die Unterscheidung von drei TVB-Allelen (TVB*S1, TVB*S3 und TVB*R). Wir haben einen PCR-RFLP-Test unter Verwendung der beiden SNPs entwickelt, um die drei beobachteten allelen Haplotypen zu bestimmen und die sechs möglichen aus diesen drei Haplotypen bestehenden TVB-Genotypen in definierten Hühner-Laborstämmen zu identifizieren. Ein weiterer potentieller alleler Haplotyp und vier Genotypen werden ebenfalls kurz besprochen. Hühnerküken von für verschiedene TVB-Allelen heterozygoten Elterntieren wurden mit Rous Sarkom-Viren (RSVs) der Subgruppen ALVB und ALVE infiziert, um Wing-Web-Tumoren zu induzieren. Bei diesen Hühnern, deren Genotyp mit Hilfe des neu entwickelten PCR-RFLP-Tests bestimmt wurde, wurde das Auftreten von Tumoren ermittelt. Bedeutsamerweise waren die Hühner, die mit dem Test als TVB*S3/*S3 typisiert wurden, nur resistent gegen die ALVE-Infektion und die als TVB*R/R typisierten waren resistent sowohl gegen ALVE Und ALVB. Ferner entwickelten die Mehrzahl der Hühner mit dem empfänglichen TVB*S1/- -Genotyp einen Tumor. Dieser PCR-RFLP-Test ermöglicht eine relativ schnelle Bestimmung aller sechs vorkommenden TVB-Genotypen durch Abgrenzung der TVB*S1-, TVB*S3- und TVB*R-Allele. Dieses nicht infektiöse Testverfahren sollte hinsichtlich seiner Eignung für die Selektion und Zucht kommerzieller Hühner auf genetische Resistenz gegen Infektionen mit ALVB, ALVD und ALVE weitergehend untersucht werden. Desarrollo y validación de una técnica de PCR-RFLP para evaluar los haplotipos TVB codificantes de receptores para los subgrupos B y E de virus de leucosis aviar en aves White Leghorn El receptor celular del subgrupo B del virus de leucosis aviar (ALVB) es codificado por un gen en el locus (TVB) del virus tumoral B. Los alelos TVB codifican para los receptores específicos permitiendo la infección por virus exógenos ALVB o ALV subgrupo D (ALVD) así como los ALV endógenos del subgrupo E (ALVE) y, por lo tanto, la susceptibilidad es dominante respecto a la resistencia. Se han descrito dos únicos polimorfismos de nucleótidos (SNP) en el locus TVB y se distinguen tres alelos TVB (TVB*S1, TVB*S3 y TVB*R). En el presente trabajo se ha desarrollado una técnica de PCR-RFLP utilizando los dos SNPs para definir tres haplotipos alélicos observados para identificar lo seis posibles genotipos de TVB que consistían en los tres haplotipos definidos en razas de laboratorio de pollos. Un haplotipo alélico potencial adicional y cuatro genotipos también se discuten brevemente. Pollos de padres heterocigotos de diferentes alelos TVB fueron infectados con virus del sarcoma de Rous (RSVs) de los subgrupos ALVB y ALVE para inducir tumores vía inoculación al ala. Se determinó la incidencia de tumores entre los pollos de los genotipos determinados por este nuevo método de PCR-RFLP. Es remarcable que los pollos tipificados con esta técnica como TVB*S3/*S3 fueron resistentes únicamente a la infección por ALVE, mientras que los TVB*R/*R fueron resistentes tanto a los ALVE como a los ALVB. Además, una vasta mayoría de pollos con los genotipos susceptibles TVB*S1/- desarrollaron un tumor. Este método de PCR-RFLP permite una determinación rápida de los seis genotipos TVB anticipados en cepas experimentales de pollos con segregación para los alelos TVB*S1, TVB*S3, y TVB*R. Esa técnica no infecciosa debe ser evaluada con más detalle para saber si se puede seleccionar y cruzar las diferentes razas comerciales de pollos para mejorar la resistencia genética a las infecciones por ALVB, ALVD y ALVE.

The tumour virus B (TVB) locus encodes cellular receptors mediating infection by three subgroups of avian leukosis virus (B, D, and E). Three major alleles, TVB*S1, TVB*S3, and TVB*R, have been described. TVB*S1 encodes a cellular receptor mediating infection of subgroups B, D, and E. TVB*S3 encodes the receptor for two subgroups, B and D, and TVB*R encodes a dysfunctional receptor that does not permit infection by any of the subgroups, B, D, or E. Genetic diversity at the TVB locus of chickens was investigated in both layer and broiler commercial pure lines and laboratory lines. Genotyping assays were developed for both medium-throughput and high-throughput analysis. Of the 36 broiler lines sampled, 14 were fixed for the susceptible allele TVB*S1. Across all broiler lines, 83% of chickens were typed as TVB*S1/*S1, 3% as TVB*R/*R, and 14% as TVB*S1/*R. In the egg-layer lines, five of the 16 tested were fixed for TVB*S1/*S1. About 44% of egg-layers were typed as TVB*S1/*S1, 15% as TVB*R/*R, with the rest segregating for two or three of the alleles. In the laboratory chickens, 60% were fixed for TVB*S1/*S1, 6% for TVB*S3/*S3, 14% for TVB*R/*R, and the rest were heterozygotes (TVB*S1/*S3 or TVB*S1/*R). All commercial pure lines examined in this study carry the TVB*S1 allele that sustains the susceptibility to avian leukosis viruses B, D, and E. More importantly, the TVB*R allele was identified in multiple populations, thus upholding the opportunities for genetic improvement through selection.