Laurence Flori

The genome response to artificial selection: a case study in dairy cattle.

Dairy cattle breeds have been subjected over the last fifty years to intense artificial selection towards improvement of milk production traits. In this study, we performed a whole genome scan for differentiation using 42,486 SNPs in the three major French dairy cattle breeds (Holstein, Normande and Montbéliarde) to identify the main physiological pathways and regions which were affected by this selection. After analyzing the population structure, we estimated FST within and across the three breeds for each SNP under a pure drift model. We further considered two different strategies to evaluate the effect of selection at the genome level. First, smoothing FST values over each chromosome with a local variable bandwidth kernel estimator allowed identifying 13 highly significant regions subjected to strong and/or recent positive selection. Some of them contained genes within which causal variants with strong effect on milk production traits (GHR) or coloration (MC1R) have already been reported. To go further in the interpretation of the observed signatures of selection we subsequently concentrated on the annotation of differentiated genes defined according to the FST value of SNPs localized close or within them. To that end we performed a comprehensive network analysis which suggested a central role of somatotropic and gonadotropic axes in the response to selection. Altogether, these observations shed light on the antagonism, at the genome level, between milk production and reproduction traits in highly producing dairy cows.

A whole genome Bayesian scan for adaptive genetic divergence in West African cattle

BackgroundThe recent settlement of cattle in West Africa after several waves of migration from remote centres of domestication has imposed dramatic changes in their environmental conditions, in particular through exposure to new pathogens. West African cattle populations thus represent an appealing model to unravel the genome response to adaptation to tropical conditions. The purpose of this study was to identify footprints of adaptive selection at the whole genome level in a newly collected data set comprising 36,320 SNPs genotyped in 9 West African cattle populations.ResultsAfter a detailed analysis of population structure, we performed a scan for SNP differentiation via a previously proposed Bayesian procedure including extensions to improve the detection of loci under selection. Based on these results we identified 53 genomic regions and 42 strong candidate genes. Their physiological functions were mainly related to immune response (MHC region which was found under strong balancing selection, CD79A, CXCR4, DLK1, RFX3, SEMA4A, TICAM1 and TRIM21), nervous system (NEUROD6, OLFM2, MAGI1, SEMA4A and HTR4) and skin and hair properties (EDNRB, TRSP1 and KRTAP8-1).ConclusionThe main possible underlying selective pressures may be related to climatic conditions but also to the host response to pathogens such as Trypanosoma(sp). Overall, these results might open the way towards the identification of important variants involved in adaptation to tropical conditions and in particular to resistance to tropical infectious diseases.

Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response

BackgroundDesigning sustainable animal production systems that better balance productivity and resistance to disease is a major concern. In order to address questions related to immunity and resistance to disease in pig, it is necessary to increase knowledge on its immune system and to produce efficient tools dedicated to this species.ResultsA long-oligonucleotide-based chip referred to as SLA-RI/NRSP8-13K was produced by combining a generic set with a newly designed SLA-RI set that targets all annotated loci of the pig major histocompatibility complex (MHC) region (SLA complex) in both orientations as well as immunity genes outside the SLA complex.The chip was used to study the immune response of pigs following stimulation of porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) or a mixture of phorbol myristate acetate (PMA) and ionomycin for 24 hours. Transcriptome analysis revealed that ten times more genes were differentially expressed after PMA/ionomycin stimulation than after LPS stimulation. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than of B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 (tumor necrosis factor receptor superfamily, member 9) genes. In addition, a very intense repression of THBS1 (thrombospondin 1) was observed. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing. Repression of MHC class II genes was observed after both stimulations. Our results provide preliminary data suggesting that antisense transcripts mapping to the SLA complex may have a role during immune response.ConclusionThe SLA-RI/NRSP8-13K chip was found to accurately decipher two distinct immune response activations of PBMCs indicating that it constitutes a valuable tool to further study immunity and resistance to disease in pig. The transcriptome analysis revealed specific and common features of the immune responses depending on the stimulation agent that increase knowledge on pig immunity.

Gene-Expression Profiling Discriminates between Cerebral Malaria (CM)–Susceptible Mice and CM-Resistant Mice

The development of cerebral malaria (CM) in mice with Plasmodium berghei ANKA infection is under genetic control. Brain gene-expression patterns were investigated in well-defined genetically CM-resistant (CM-R; BALB/c and DBA/2) and CM-susceptible (CM-S; C57BL/6 and CBA/J) mice by use of cDNA microarrays. By combining transcriptional profiling with rigorous statistical methods and cluster analysis, we identified a set of 69 genes that perfectly discriminated between mouse strains and between CM-R and CM-S mice. The analysis of gene ontological terms revealed that the genes that clustered and were related to susceptibility to CM preferentially belonged to some biological process classes, such as those pertaining to immune responses. Using a false discovery rate of 5% and the Welch t test, we identified 31 genes with consistent differential expression between CM-R and CM-S mice. These data indicate that microarray analysis may be useful for identification of candidate genes that are potentially responsible for resistance or susceptibility to mouse CM and suggest that candidate genes identified in mice could be specifically tested in humans for an association with disease severity.

Linkage and association between Plasmodium falciparum blood infection levels and chromosome 5q31-q33

We have previously mapped a locus controlling Plasmodium falciparum blood infection levels (PFBI) to chromosome 5q31–q33. We genotyped 19 microsatellite markers on chromosome 5q31–q33 in a new sample of 44 pedigrees comprising 84 nuclear families and 292 individuals living in a P. falciparum endemic area. Using a nonparametric multipoint variance-component approach (by GENEHUNTER), we evidenced a peak of linkage close to D5S636 (P=0.0069), with a heritability of 0.46. Using a variance-component method for linkage-disequilibrium mapping of quantitative traits (by QTDT) and the Bonferroni correction for multiple testing, we further detected allelic association in the presence of linkage between blood infection levels and D5S487 (P=6 × 10−5; Pc=0.0011), which is located on the distal part of the peak. These results confirm the importance of chromosome 5q31–q33 in the genetic control of PFBI levels.

Immunity Traits in Pigs: Substantial Genetic Variation and Limited Covariation

Background Increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. As resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (ITs). Our underlying hypothesis is that levels of ITs with no focus on specific pathogens define an individuals immunocompetence and thus predict response to pathogens in general. Since variation in ITs depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. In this report, we present a large genetic survey of innate and adaptive ITs in pig families bred in the same environment. Methodology/Principal Findings Fifty four ITs were studied on 443 Large White pigs vaccinated against Mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (PCA) and genetic parameter estimation. ITs include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (IFNα, TNFα, IL6, IL8, IL12, IFNγ, IL2, IL4, IL10), phagocytosis and lymphocyte proliferation. While six ITs had heritabilities that were weak or not significantly different from zero, 18 and 30 ITs had moderate (0.10.4) heritability values, respectively. Phenotypic and genetic correlations between ITs were weak except for a few traits that mostly include cell subsets. PCA revealed no cluster of innate or adaptive ITs. Conclusions/Significance Our results demonstrate that variation in many innate and adaptive ITs is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. A limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive ITs. Our data provide a genetic framework for choosing ITs to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits.

Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection

BackgroundTranscriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.ResultsAn increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection.ConclusionOur results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

TNF as a malaria candidate gene: polymorphism-screening and family-based association analysis of mild malaria attack and parasitemia in Burkina Faso

We have previously obtained strong evidence for linkage of mild malaria attack to the MHC region, with a peak close to the tumor necrosis factor (TNF) gene. We screened, for polymorphisms, the entire TNF gene in the same sample of 34 families comprising 197 individuals living in a Plasmodium falciparum endemic area and we found 17 polymorphisms. In a longitudinal study, we investigated whether the 11 most frequent and informative polymorphisms were associated with mild malaria attack and maximum parasitemia, which was the highest parasitemia in each individual over 2 years. Mild malaria attack and maximum parasitemia were positively correlated. Transmission disequilibrium tests showed nominal evidence for association between TNF-1031, TNF-308, TNF851 and TNF1304 polymorphisms, and mild malaria attack on the one hand, and between TNF-238, TNF851 and TNF1304 polymorphisms, and maximum parasitemia on the other hand. After accounting for multiple tests, we confirmed the association of TNF-238 with maximum parasitemia and the association of TNF1304 and TNF851 with maximum parasitemia and mild malaria attack. The association tests with mild malaria attack suggest a moderate effect of TNF-308 polymorphism. In conclusion, our study suggests that several TNF variants may be part of the genetic determinants for maximum parasitemia and/or mild malaria attack.

Co-expression of host and viral microRNAs in porcine dendritic cells infected by the pseudorabies virus.

MicroRNAs are small non-coding RNAs approximately 22 nt long that modulate gene expression in animals and plants. It has been recently demonstrated that herpesviruses encode miRNAs to control the post-transcriptional regulation of expression from their own genomes and possibly that of their host, thus adding an additional layer of complexity to the physiological cross-talk between host and pathogen. The present study focussed on the interactions between porcine dendritic cells (DCs) and the Pseudorabies virus (PRV), an alpha-herpesvirus causing Aujeszkys disease in pigs. A catalogue of porcine and viral miRNAs, expressed eight hours post-infection, was established by deep sequencing. An average of 2 million reads per sample with a size of 21–24 nucleotides was obtained from six libraries representing three biological replicates of infected and mock-infected DCs. Almost 95% of reads mapped to the draft pig genome sequence and pig miRNAs previously annotated in dedicated databases were detected by sequence alignment. In silico prediction allowed the identification of unknown porcine as well as of five miRNAs transcribed by the Large Latency Transcript (LLT) of PRV. The gene target prediction of the viral miRNAs and the Ingenuity Pathway Analysis of differentially expressed pig miRNAs were conducted to contextualize the identified small RNA molecules and functionally characterize their involvement in the post-transcriptional regulation of gene expression. The results support a role for PRV miRNAs in the maintenance of the host cell latency state through the down-regulation of immediate-early viral genes which is similar to other herpesviruses. The differentially expressed swine miRNAs identified a unique network of target genes with highly significant functions in the development and function of the nervous system and in infectious mechanisms, suggesting that the modulation of both host and viral miRNAs is necessary for the establishment of PRV latency.

Influence of Carriage of Hemoglobin AS and the Fcγ Receptor IIa–R131 Allele on Levels of Immunoglobulin G2 Antibodies to Plasmodium falciparum Merozoite Antigens in Gabonese Children

BACKGROUND To extend our previous findings showing an imbalanced distribution of immunoglobulin G2 (IgG2) antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2) and a higher frequency of infection with multiple P. falciparum strains in Gabonese children with sickle cell trait (hemoglobin AS), human Fc gamma receptor (Fc gamma R) IIa (CD32) polymorphism and the rate of in vitro invasion of red blood cells (RBCs) from subjects with either hemoglobin AA or AS by multiple P. falciparum strains were investigated. METHODS Fc gamma RIIa mutation at amino acid position 131 (arginine or histidine) was detected by polymerase chain reaction, and in vitro cultures for parasites were used to assess the invasion rate. RESULTS Fc gamma RIIa polymorphism is normally distributed in this population, with no preferential carriage by children with hemoglobin AS. Lower levels of IgG2 subclass antibodies to MSP2 peptides were independently associated with the Fc gamma RIIa-R131 allele and with carriage of hemoglobin AS. Our data suggest that IgG3 antibody responses to MSP2 epitopes could be exacerbated by lower IgG2 levels in children with hemoglobin AS. CONCLUSIONS The higher rate of invasion of RBCs in the presence of multiple strains may indicate that several invasion pathways are solicited simultaneously, and the longer persistence of ring forms in RBCs from the subjects with hemoglobin AS might reflect a slower multiplication phase, leading to a longer circulation and enhanced phagocytosis of these nonpathogenic parasite forms. This may contribute to the protection against P. falciparum malaria observed in children with hemoglobin AS.