Understanding bovine embryo elongation: a transcriptomic study of trophoblastic vesicles.

Background: During the process of elongation, the embryo increases in size within the uterus, while the extra-embryonic tissues (EETs) develop and differentiate in preparation for implantation. As it grows, the ovoid embryo transforms into a tubular form first and then a filamentous form. This process is directed by numerous genes and pathways, the expression of which may be altered in the case of developmental irregularities such as when the conceptus is shorter than expected or when the embryo develops after splitting. In bovines, efforts to understand the molecular basis of elongation have employed trophoblastic vesicles (TVs)-short tubular EET pieces that lack an embryo-which also elongate in vivo. To date, however, we lack molecular analyses of TVs at the ovoid or filamentous stages that might shed light on the expression changes involved. Methods: Following in vivo development, we collected bovine conceptuses from the ovoid (D12) to filamentous stages (D18), sectioned them into small pieces with or without their embryonic disc (ED), and then, transferred them to a receptive bovine uterus to assess their elongation abilities. We also grew spherical blastocysts in vitro up to D8 and subjected them to the same treatment. Then, we assessed the differences in gene expression between different samples and fully elongating controls at different stages of elongation using a bovine array (10 K) and an extended qPCR array comprising 224 genes across 24 pathways. Results: In vivo, TVs elongated more or less depending on the stage at which they had been created and the time spent in utero. Their daily elongation rates differed from control EET, with the rates of TVs sometimes resembling those of earlier-stage EET. Overall, the molecular signatures of TVs followed a similar developmental trajectory as intact EET from D12-D18. However, within each stage, TVs and intact EET displayed distinct expression dynamics, some of which were shared with other short epithelial models. Conclusion: Differences between TVs and EET likely result from multiple factors, including a reduction in the length and signaling capabilities of TVs, delayed elongation from inadequate uterine signals, and modified crosstalk between the conceptus and the uterus. These findings confirm that close coordination between uterine, embryonic, and extra-embryonic tissues is required to orchestrate proper elongation and, based on the partial differentiation observed, raise questions about the presence/absence of certain developmental cues or even their asynchronies.

Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing.

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis. The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis, and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Comparative analysis of the caecal tonsil transcriptome in two chicken lines experimentally infected with Salmonella Enteritidis.

Managing Salmonella enterica Enteritidis (SE) carriage in chicken is necessary to ensure human food safety and enhance the economic, social and environmental sustainability of chicken breeding. Salmonella can contaminate poultry products, causing human foodborne disease and economic losses for farmers. Both genetic selection for a decreased carriage and gut microbiota modulation strategies could reduce Salmonella propagation in farms. Two-hundred and twenty animals from the White Leghorn inbred lines N and 61 were raised together on floor, infected by SE at 7 days of age, transferred into isolators to prevent oro-fecal recontamination and euthanized at 12 days post-infection. Caecal content DNA was used to measure individual Salmonella counts (ISC) by droplet digital PCR. A RNA sequencing approach was used to measure gene expression levels in caecal tonsils after infection of 48 chicks with low or high ISC. The analysis between lines identified 7516 differentially expressed genes (DEGs) corresponding to 62 enriched Gene Ontology (GO) Biological Processes (BP) terms. A comparison between low and high carriers allowed us to identify 97 DEGs and 23 enriched GO BP terms within line 61, and 1034 DEGs and 288 enriched GO BP terms within line N. Among these genes, we identified several candidate genes based on their putative functions, including FUT2 or MUC4, which could be involved in the control of SE infection, maybe through interactions with commensal bacteria. Altogether, we were able to identify several genes and pathways associated with differences in SE carriage level. These results are discussed in relation to individual caecal microbiota compositions, obtained for the same animals in a previous study, which may interact with host gene expression levels for the control of the caecal SE load.

Differences in caecal microbiota composition and Salmonella carriage between experimentally infected inbred lines of chickens.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major causes of human foodborne intoxication resulting from consumption of contaminated poultry products. Genetic selection of animals that are more resistant to Salmonella carriage and modulation of the gut microbiota are two promising ways to decrease individual Salmonella carriage. The aims of this study were to identify the main genetic and microbial factors that control the level of Salmonella carriage in chickens (Gallus gallus) under controlled experimental conditions. Two-hundred and forty animals from the White Leghorn inbred lines N and 61 were infected by SE at 7 days of age. After infection, animals were kept in isolators to reduce recontamination of birds by Salmonella. Caecal contents were sampled at 12 days post-infection and used for DNA extraction. Microbiota DNA was used to measure individual counts of SE by digital PCR and to determine the bacterial taxonomic composition, using a 16S rRNA gene high-throughput sequencing approach. RESULTS: Our results confirmed that the N line is more resistant to Salmonella carriage than the 61 line, and that intra-line variability is higher for the 61 line. Furthermore, the 16S analysis showed strong significant differences in microbiota taxonomic composition between the two lines. Among the 617 operational taxonomic units (OTU) observed, more than 390 were differentially abundant between the two lines. Furthermore, within the 61 line, we found a difference in the microbiota taxonomic composition between the high and low Salmonella carriers, with 39 differentially abundant OTU. Using metagenome functional prediction based on 16S data, several metabolic pathways that are potentially associated to microbiota taxonomic differences (e.g. short chain fatty acids pathways) were identified between high and low carriers. CONCLUSIONS: Overall, our findings demonstrate that the caecal microbiota composition differs between genetic lines of chickens. This could be one of the reasons why the investigated lines differed in Salmonella carriage levels under experimental infection conditions.

Draft Genome Sequences of 142 Mycobacterium avium subsp. paratuberculosis Strains Isolated from Naturally Infected Dairy Cattle.

Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 M. avium subsp. paratuberculosis strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.

Links between fecal microbiota and the response to vaccination against influenza A virus in pigs.

This study describes the associations between fecal microbiota and vaccine response variability in pigs, using 98 piglets vaccinated against the influenza A virus at 28 days of age (D28) with a booster at D49. Immune response to the vaccine is measured at D49, D56, D63, and D146 by serum levels of IAV-specific IgG and assays of hemagglutination inhibition (HAI). Analysis of the pre-vaccination microbiota characterized by 16S rRNA gene sequencing of fecal DNA reveals a higher vaccine response in piglets with a richer microbiota, and shows that 23 operational taxonomic units (OTUs) are differentially abundant between high and low IAV-specific IgG producers at D63. A stronger immune response is linked with OTUs assigned to the genus Prevotella and family Muribaculaceae, and a weaker response is linked with OTUs assigned to the genera Helicobacter and Escherichia-Shigella. A set of 81 OTUs accurately predicts IAV-specific IgG and HAI titer levels at all time points, highlighting early and late associations between pre-vaccination fecal microbiota composition and immune response to the vaccine.

The fecal microbiota of piglets during weaning transition and its association with piglet growth across various farm environments.

This study describes the fecal microbiota from piglets reared in different living environments during the weaning transition, and presents the characteristics of microbiota associated with good growth of piglets after weaning. Fecal samples were collected pre- (d26) and post-weaning (d35) from 288 male piglets in 16 conventional indoor commercial farms located in the West of France. The changes one week after weaning on the most abundant microbial families was roughly the same in all farms: alpha diversity increased, the relative abundance of Bacteroidaceae (-61%), Christensenellaceae (-35%), Enterobacteriaceae (-42%), and Clostridiaceae (-32%) decreased, while the relative abundance of Prevotellaceae (+143%) and Lachnospiraceae (+21%) increased. Among all the collected samples, four enterotypes that were ubiquitous in all farms were identified. They could be discriminated by their respective relative abundances of Prevotella, Faecalibacterium, Roseburia, and Lachnospira, and likely corresponded to a gradual maturational shift from pre- to post-weaning microbiota. The rearing environment influenced the frequency of enterotypes, as well as the relative abundance of 6 families at d26 (including Christensenellaceae and Lactobacillaceae), and of 21 families at d35. In all farms, piglets showing the highest relative growth rate during the first three weeks after weaning, which were characterized as more robust, had a higher relative abundance of Bacteroidetes, a lower relative abundance of Proteobacteria, and showed a greater increase in Prevotella, Coprococcus, and Lachnospira in the post-weaning period. This study revealed the presence of ubiquitous enterotypes among the farms of this study, reflecting maturational stages of microbiota from a young suckling to an older cereal-eating profile. Despite significant variation in the microbial profile between farms, piglets whose growth after weaning was less disrupted were, those who had reached the more mature phenotype characterized by Prevotella the fastest.

A Probiotic Mixture Induces Anxiolytic- and Antidepressive-Like Effects in Fischer and Maternally Deprived Long Evans Rats.

A role of the gut microbiota in psychiatric disorders is supported by a growing body of literature. The effects of a probiotic mixture of four bacterial strains were studied in two models of anxiety and depression, naturally stress-sensitive Fischer rats and Long Evans rats subjected to maternal deprivation. Rats chronically received either the probiotic mixture (1.109 CFU/day) or the vehicle. Anxiety- and depressive-like behaviors were evaluated in several tests. Brain monoamine levels and gut RNA expression of tight junction proteins (Tjp) and inflammatory markers were quantified. The gut microbiota was analyzed in feces by 16S rRNA gene sequencing. Untargeted metabolite analysis reflecting primary metabolism was performed in the cecal content and in serum. Fischer rats treated with the probiotic mixture manifested a decrease in anxiety-like behaviors, in the immobility time in the forced swimming test, as well as in levels of dopamine and its major metabolites, and those of serotonin metabolites in the hippocampus and striatum. In maternally deprived Long Evans rats treated with the probiotic mixture, the number of entries into the central area in the open-field test was increased, reflecting an anxiolytic effect. The probiotic mixture increased Tjp1 and decreased Ifnγ mRNA levels in the ileum of maternally deprived rats. In both models, probiotic supplementation changed the proportions of several Operational Taxonomic Units (OTU) in the gut microbiota, and the levels of certain cecal and serum metabolites were correlated with behavioral changes. Chronic administration of the tested probiotic mixture can therefore beneficially affect anxiety- and depressive-like behaviors in rats, possibly owing to changes in the levels of certain metabolites, such as 21-deoxycortisol, and changes in brain monoamines.

Discovery of Predictors of Mycoplasma hyopneumoniae Vaccine Response Efficiency in Pigs: 16S rRNA Gene Fecal Microbiota Analysis.

The gut microbiota comprises a large and diverse community of bacteria that play a significant role in swine health. Indeed, there is a tight association between the enteric immune system and the overall composition and richness of the microbiota, which is key in the induction, training and function of the host immunity, and may therefore, influence the immune response to vaccination. Using vaccination against Mycoplasma hyopneumoniae (M. hyo) as a model, we investigated the potential of early-life gut microbiota in predicting vaccine response and explored the post-vaccination dynamics of fecal microbiota at later time points. At 28 days of age (0 days post-vaccination; dpv), healthy piglets were vaccinated, and a booster vaccine was administered at 21 dpv. Blood samples were collected at 0, 21, 28, 35, and 118 dpv to measure M. hyo-specific IgG levels. Fecal samples for 16S rRNA gene amplicon sequencing were collected at 0, 21, 35, and 118 dpv. The results showed variability in antibody response among individual pigs, whilst pre-vaccination operational taxonomic units (OTUs) primarily belonging to Prevotella, [Prevotella], Anaerovibrio, and Sutterella appeared to best-predict vaccine response. Microbiota composition did not differ between the vaccinated and non-vaccinated pigs at post-vaccination time points, but the time effect was significant irrespective of the animals' vaccination status. Our study provides insight into the role of pre-vaccination gut microbiota composition in vaccine response and emphasizes the importance of studies on full metagenomes and microbial metabolites aimed at deciphering the role of specific bacteria and bacterial genes in the modulation of vaccine response.

Late weaning is associated with increased microbial diversity and Faecalibacterium prausnitzii abundance in the fecal microbiota of piglets.

BACKGROUND: In pig production systems, weaning is a crucial period characterized by nutritional, environmental, and social stresses. Piglets transition from a milk-based diet to a solid, more complex plant-based diet, and their gut physiology must adapt accordingly. It is well established that piglets weaned later display improved health, better wean-to-finish growth performance, and lower mortality rates. The aim of this study was to evaluate the impact of weaning age on fecal microbiota diversity and composition in piglets. Forty-eight Large White piglets were divided into 4 groups of 12 animals that were weaned at different ages: 14 days (early weaning), 21 days (a common weaning age in intensive pig farming), 28 days (idem), and 42 days (late weaning). Microbiota composition was assessed in each group by sequencing the 16S rRNA gene using fecal samples taken on the day of weaning, 7 days later, and at 60 days of age. RESULTS: In each group, there were significant differences in fecal microbiota composition before and after weaning (p < 0.05), confirming that weaning can drastically change the gut microbiota. Microbiota diversity was positively correlated with weaning age: microbial alpha diversity and richness were higher in piglets weaned at 42 days of age both on the day of weaning and 7 days later. The abundance of Faecalibacterium prausnitzii operational taxonomic units (OTUs) was also higher in piglets weaned at 42 days of age. CONCLUSIONS: Overall, these results show that late weaning increased gut microbiota diversity and the abundance of F. prausnitzii, a microorganism with positive effects in humans. Piglets might thus derive a competitive advantage from later weaning because they have more time to accumulate a higher diversity of potentially beneficial microbes prior to the stressful and risky weaning period.

Draft Anaplasma phagocytophilum Genome Sequences from Five Cows, Two Horses, and One Roe Deer Collected in Europe.

Anaplasma phagocytophilum is a zoonotic tick-borne intracellular bacterium responsible for granulocytic anaplasmosis. As it is difficult to isolate and cultivate, only 20 A. phagocytophilum genomes have been sequenced to date. Here, we present eight A. phagocytophilum genome sequences obtained using alternative approaches based on sequence capture technology.

The ß-importin KAP8 (Pse1/Kap121) is required for nuclear import of the cellulase transcriptional regulator XYR1, asexual sporulation and stress resistance in Trichoderma reesei.

The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes, and serves as a prime model for their genetic regulation. Most of its (hemi-)cellulolytic enzymes are obligatorily dependent on the transcriptional activator XYR1. Here, we investigated the nucleo-cytoplasmic shuttling mechanism that transports XYR1 across the nuclear pore complex. We identified 14 karyopherins in T. reesei, of which eight were predicted to be involved in nuclear import, and produced single gene-deletion mutants of all. We found KAP8, an ortholog of Aspergillus nidulans KapI, and Saccharomyces cerevisiae Kap121/Pse1, to be essential for nuclear recruitment of GFP-XYR1 and cellulase gene expression. Transformation with the native gene rescued this effect. Transcriptomic analyses of Δkap8 revealed that under cellulase-inducing conditions 42 CAZymes, including all cellulases and hemicellulases known to be under XYR1 control, were significantly down-regulated. Δkap8 strains were capable of forming fertile fruiting bodies but exhibited strongly reduced conidiation both in light and darkness, and showed enhanced sensitivity towards abiotic stress, including high osmotic pressure, low pH and high temperature. Together, these data underscore the significance of nuclear import of XYR1 in cellulase and hemicellulase gene regulation in T. reesei, and identify KAP8 as the major karyopherin required for this process.

Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle.

The 1000 bull genomes project supports the goal of accelerating the rates of genetic gain in domestic cattle while at the same time considering animal health and welfare by providing the annotated sequence variants and genotypes of key ancestor bulls. In the first phase of the 1000 bull genomes project, we sequenced the whole genomes of 234 cattle to an average of 8.3-fold coverage. This sequencing includes data for 129 individuals from the global Holstein-Friesian population, 43 individuals from the Fleckvieh breed and 15 individuals from the Jersey breed. We identified a total of 28.3 million variants, with an average of 1.44 heterozygous sites per kilobase for each individual. We demonstrate the use of this database in identifying a recessive mutation underlying embryonic death and a dominant mutation underlying lethal chrondrodysplasia. We also performed genome-wide association studies for milk production and curly coat, using imputed sequence variants, and identified variants associated with these traits in cattle.

Novel insights into the bovine polled phenotype and horn ontogenesis in Bovidae.

Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.

A 3.7 Mb deletion encompassing ZEB2 causes a novel polled and multisystemic syndrome in the progeny of a somatic mosaic bull.

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

A newly described bovine type 2 scurs syndrome segregates with a frame-shift mutation in TWIST1.

The developmental pathways involved in horn development are complex and still poorly understood. Here we report the description of a new dominant inherited syndrome in the bovine Charolais breed that we have named type 2 scurs. Clinical examination revealed that, despite a strong phenotypic variability, all affected individuals show both horn abnormalities similar to classical scurs phenotype and skull interfrontal suture synostosis. Based on a genome-wide linkage analysis using Illumina BovineSNP50 BeadChip genotyping data from 57 half-sib and full-sib progeny, this locus was mapped to a 1.7 Mb interval on bovine chromosome 4. Within this region, the TWIST1 gene encoding a transcription factor was considered as a strong candidate gene since its haploinsufficiency is responsible for the human Saethre-Chotzen syndrome, characterized by skull coronal suture synostosis. Sequencing of the TWIST1 gene identified a c.148_157dup (p.A56RfsX87) frame-shift mutation predicted to completely inactivate this gene. Genotyping 17 scurred and 20 horned founders of our pedigree as well as 48 unrelated horned controls revealed a perfect association between this mutation and the type 2 scurs phenotype. Subsequent genotyping of 32 individuals born from heterozygous parents showed that homozygous mutated progeny are completely absent, which is consistent with the embryonic lethality reported in Drosophila and mouse suffering from TWIST1 complete insufficiency. Finally, data from previous studies on model species and a fine description of type 2 scurs symptoms allowed us to propose different mechanisms to explain the features of this syndrome. In conclusion, this first report on the identification of a potential causal mutation affecting horn development in cattle offers a unique opportunity to better understand horn ontogenesis.