Using genome-wide SNP data, we calculated genomic inbreeding coefficients (FROH > 1  Mb , FROH > 2 Mb , FROH > 8 Mb and FROH > 16 Mb ) derived from runs of homozygosity (ROH) of different lengths (>1, >2, >8 and > 16 Mb) as well as from levels of homozygosity (FHOM ). We compared these values of inbreeding coefficients with those calculated from pedigrees (FPED ) of 1422 bulls comprising Brown Swiss (304), Fleckvieh (502), Norwegian Red (499) and Tyrol Grey (117) cattle breeds. For all four breeds, population inbreeding levels estimated by the genomic inbreeding coefficients FROH > 8 Mb and FROH > 16 Mb were similar to the levels estimated from pedigrees. The lowest values were obtained for Fleckvieh (FPED  = 0.014, FROH > 8 Mb  = 0.019 and FROH > 16 Mb  = 0.008); the highest, for Brown Swiss (FPED  = 0.048, FROH > 8 Mb  = 0.074 and FROH > 16 Mb  = 0.037). In contrast, inbreeding estimates based on the genomic coefficients FROH > 1 Mb and FROH > 2 Mb were considerably higher than pedigree-derived estimates. Standard deviations of genomic inbreeding coefficients were, on average, 1.3-1.7-fold higher than those obtained from pedigrees. Pearson correlations between genomic and pedigree inbreeding coefficients ranged from 0.50 to 0.62 in Norwegian Red (lowest correlations) and from 0.64 to 0.72 in Tyrol Grey (highest correlations). We conclude that the proportion of the genome present in ROH provides a good indication of inbreeding levels and that analysis based on ROH length can indicate the relative amounts of autozygosity due to recent and remote ancestors.

Chickens from two inbred lines selected for high (L10H) or low (L10L) mannose-binding lectin (MBL) serum concentrations were infected with infectious bronchitis virus (IBV), and innate as well as adaptive immunological parameters were measured throughout the experimental period. Chickens with high MBL serum concentrations were found to have less viral load in the trachea than chickens with low MBL serum concentrations indicating that these chickens were less severely affected by the infection. This study is the first to show that MBL expression is present in the lungs of healthy chickens and that the expression is upregulated at days 3 postinfection (p.i.) in L10H chickens. Furthermore, in the liver of infected chickens, the MBL expression was upregulated at day 7 p.i., despite the fact that the MBL serum concentrations were decreased below baseline at that time point. The number of TCRcd + CD8a + cells in the blood of noninfected chickens increased from week 0 to 3 p.i. However, the number of cells was higher in L10H chickens than in L10L chickens throughout the experiment. No increase was observed in the number of TCRcd + CD8a + cells in the blood of the infected L10H and L10L chickens. The numbers of B cells at week 3 p.i. were higher for noninfected L10L chickens than for the other chickens. No differences were observed between the infected and noninfected L10H chickens or between the infected L10H and L10L chickens. Furthermore, at week 3 p.i., the number of monocytes was higher in infected and noninfected L10H chickens than in the infected and noninfected L10L chickens. Thus, these results indicate that MBL is produced locally and may be involved in the regulation of the cellular immune response after an IBV infection. However, MBL did not appear to influence the humoral immune response after IBV infection in this study.

BackgroundCoccidiosis is the most common and costly disease in the poultry industry and is caused by protozoans of the Eimeria genus. The current control of coccidiosis, based on the use of anticoccidial drugs and vaccination, faces serious obstacles such as drug resistance and the high costs for the development of efficient vaccines, respectively. Therefore, the current control programs must be expanded with complementary approaches such as the use of genetics to improve the host response to Eimeria infections. Recently, we have performed a large-scale challenge study on Cobb500 broilers using E. maxima for which we investigated variability among animals in response to the challenge. As a follow-up to this challenge study, we performed a genome-wide association study (GWAS) to identify genomic regions underlying variability of the measured traits in the response to Eimeria maxima in broilers. Furthermore, we conducted a post-GWAS functional analysis to increase our biological understanding of the underlying response to Eimeria maxima challenge.ResultsIn total, we identified 22 single nucleotide polymorphisms (SNPs) with q value <0.1 distributed across five chromosomes. The highly significant SNPs were associated with body weight gain (three SNPs on GGA5, one SNP on GGA1 and one SNP on GGA3), plasma coloration measured as optical density at wavelengths in the range 465–510 nm (10 SNPs and all on GGA10) and the percentage of β2-globulin in blood plasma (15 SNPs on GGA1 and one SNP on GGA2). Biological pathways related to metabolic processes, cell proliferation, and primary innate immune processes were among the most frequent significantly enriched biological pathways. Furthermore, the network-based analysis produced two networks of high confidence, with one centered on large tumor suppressor kinase 1 (LATS1) and 2 (LATS2) and the second involving the myosin heavy chain 6 (MYH6).ConclusionsWe identified several strong candidate genes and genomic regions associated with traits measured in response to Eimeria maxima in broilers. Furthermore, the post-GWAS functional analysis indicates that biological pathways and networks involved in tissue proliferation and repair along with the primary innate immune response may play the most important role during the early stage of Eimeria maxima infection in broilers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12711-015-0170-0) contains supplementary material, which is available to authorized users.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

BackgroundAvian infectious bronchitis is a highly contagious disease of the upper-respiratory tract caused by infectious bronchitis virus (IBV). Understanding the molecular mechanisms involved in the interaction between innate and adaptive immune responses to IBV infection is a crucial element for further improvements in strategies to control IB. To this end, two chicken lines, selected for high (L10H line) and low (L10L line) serum concentration of mannose-binding lectin (MBL) were studied. In total, 32 birds from each line were used. Sixteen birds from each line were infected with IBV and sixteen were left uninfected. Eight uninfected and infected birds from each line were euthanized at 1 and 3 weeks post infection. RNA sequencing was performed on spleen samples from all 64 birds and differential gene expression analysis was performed for four comparisons: L10L line versus L10H line for uninfected birds at weeks 1 and 3, respectively, and in the same way for infected birds. Functional analysis was performed using Gene Ontology (GO) Immune System Process terms specific for Gallus gallus.ResultsComparing uninfected L10H and L10L birds, we identified 1698 and 1424 differentially expressed (DE) genes at weeks 1 and 3, respectively. For the IBV-infected birds, 1934 and 866 DE genes were identified between the two lines at weeks 1 and 3, respectively. The two most enriched GO terms emerging from the comparison of uninfected birds between the two lines were “Lymphocyte activation involved in immune response” and “Somatic recombination of immunoglobulin genes involved in immune response” at weeks 1 and 3, respectively. When comparing IBV-infected birds between the two lines, the most enriched GO terms were “Alpha-beta T cell activation” and “Positive regulation of leukocyte activation” at weeks 1 and 3, respectively.ConclusionsHealthy birds from the two lines showed significant differences in expression profiles for subsets of adaptive and innate immunity-related genes, whereas comparison of the IBV-infected birds from the two lines showed differences in expression of immunity-related genes involved in T cell activation and proliferation. The observed transcriptome differences between the two lines indicate that selection for MBL had influenced innate as well as adaptive immunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2403-1) contains supplementary material, which is available to authorized users.

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in 2 parts: a pilot challenge on 240 animals followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 d of age. In the pilot challenge, all animals were measured for BW gain, plasma coloration, hematocrit, and rectal temperature and, in addition, a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration, and hematocrit whereas a subset of 184 animals was measured for intestinal lesion score, fecal oocyst count, blood parameters, and plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P < 0.001), oocyst count (P < 0.05), plasma coloration for the optical density values between 450 and 490 nm (P < 0.001), albumin (P < 0.001), α1-globulin (P < 0.01), α2-globulin (P < 0.001), α3-globulin (P < 0.01), and β2-globulin (P < 0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 < 0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers' response to Eimeria maxima has a strong genetic determinism, which may be improved by genetic selection.

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