Avian Evolutionary Genomics: Studies of Ficedula Flycatchers

In this thesis, different genetic tools are used to investigate both natural variation and speciation in the Ficedula flycatcher system: pied (Ficedula hypoleuca) and collared (F. albicollis) flycatchers. The molecular evolution of a gene involved in postnatal body growth, GH, has shown high degree of conservation at the mature protein between birds and mammals, whereas the variation observed in its signal peptide seems to be adaptive in pied flycatcher (I & II). Speciation is the process by which reproductive barriers to gene flow evolve between populations, and understanding the mechanisms involved in pre- and post-zygotic isolation have been investigated in Ficedula flycatchers. The Z chromosome have been suggested to be the hotspot for genes involved in speciation, thus sequencing of 13 Z-linked coding genes from the two species in allopatry and sympatry have been conducted (III). Surprisingly, the majority of Z-linked genes seemed to be highly conserved, suggesting instead a potential involvement of regulatory regions. Previous studies have shown that genes involved in hybrid fitness, female preferences and male plumage colouration are sex-linked. Hence, three pigmentation genes have been investigated: MC1R, AGRP, and TYRP1. Of these three genes, TYRP1 was identified as a strong candidate to be associated with black-brown plumage variation in sympatric populations, and hence is a strong candidate for a gene contributing to pre-zygotic isolation (IV). In sympatric areas, where pied and collared flycatchers have overlapping breeding areas, hybridization sometimes occurs leading to the production of unfit hybrids. By using a proteomic approach a novel expression pattern in hybrids was revealed compared to the parental species (V) and differentially expressed proteins subsequently identified by sequence similarity (VI). In conclusion, the Z chromosome appears to play an important role in flycatcher speciation, but probably not at the coding level. In addition the novel expression patterns might give new insights into the maladaptive hybrids.Siirretty Doriast

Comparison of the transcriptome in circulating leukocytes in early lactation between primiparous and multiparous cows provides evidence for age-related changes.

BACKGROUND: Previous studies have identified many immune pathways which are consistently altered in humans and model organisms as they age. Dairy cows are often culled at quite young ages due to an inability to cope adequately with metabolic and infectious diseases, resulting in reduced milk production and infertility. Improved longevity is therefore a desirable trait which would benefit both farmers and their cows. This study analysed the transcriptome derived from RNA-seq data of leukocytes obtained from Holstein cows in early lactation with respect to lactation number. RESULTS: Samples were divided into three lactation groups for analysis: i) primiparous (PP, n = 53), ii) multiparous in lactations 2–3 (MP 2–3, n = 121), and iii) MP in lactations 4–7 (MP > 3, n = 55). Leukocyte expression was compared between PP vs MP > 3 cows with MP 2–3 as background using DESeq2 followed by weighted gene co-expression network analysis (WGCNA). Seven modules were significantly correlated (r ≥ 0.25) to the trait lactation number. Genes from the modules which were more highly expressed in either the PP or MP > 3 cows were pooled, and the gene lists subjected to David functional annotation cluster analysis. The top three clusters from modules more highly expressed in the PP cows all involved regulation of gene transcription, particularly zinc fingers. Another cluster included genes encoding enzymes in the mitochondrial beta-oxidation pathway. Top clusters up-regulated in MP > 3 cows included the terms Glycolysis/Gluconeogenesis, C-type lectin, and Immunity. Differentially expressed candidate genes for ageing previously identified in the human blood transcriptome up-regulated in PP cows were mainly associated with T-cell function (CCR7, CD27, IL7R, CAMK4, CD28), mitochondrial ribosomal proteins (MRPS27, MRPS9, MRPS31), and DNA replication and repair (WRN). Those up-regulated in MP > 3 cows encoded immune defence proteins (LYZ, CTSZ, SREBF1, GRN, ANXA5, ADARB1). CONCLUSIONS: Genes and pathways associated with lactation number in cows were identified for the first time to date, and we found that many were comparable to those known to be associated with ageing in humans and model organisms. We also detected changes in energy utilization and immune responses in leukocytes from older cows. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-021-07977-5

Transcriptomic analysis of circulating leukocytes obtained during the recovery from clinical mastitis caused by Escherichia coli in Holstein dairy cows

SIMPLE SUMMARY: Escherichia coli is a bacterium which infects cow udders causing clinical mastitis, a potentially severe disease with welfare and economic consequences. During an infection, white blood cells (leukocytes) enter the udder to provide immune defence and assist tissue repair. We sequenced RNA derived from circulating leukocytes to investigate which genes are up- or down-regulated in dairy cows with naturally occurring cases of clinical mastitis in comparison with healthy control cows from the same farm. We also looked for genetic variations between infected and healthy cows. Blood samples were taken either EARLY (around 10 days) or LATE (after 4 weeks) during the recovery phase after diagnosis. Many genes (1090) with immune and inflammatory functions were up-regulated during the EARLY phase. By the LATE phase only 29 genes were up-regulated including six haemoglobin subunits, possibly important for the production of new red blood corpuscles. Twelve genetic variations which were associated with an increased or decreased expression of some important immune genes were identified between the infected and control cows. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite the cows having received prompt veterinary treatment, but they had largely recovered within 4 weeks. Genetic differences between cows may predispose some animals to infection. ABSTRACT: The risk and severity of clinical infection with Escherichia coli as a causative pathogen for bovine mastitis is influenced by the hosts’ phenotypic and genotypic variables. We used RNA-Seq analysis of circulating leukocytes to investigate global transcriptomic profiles and genetic variants from Holstein cows with naturally occurring cases of clinical mastitis, diagnosed using clinical symptoms and milk microbiology. Healthy lactation-matched cows served as controls (CONT, n = 6). Blood samples were collected at two time periods during the recovery phase post diagnosis: EARLY (10.3 ± 1.8 days, n = 6) and LATE (46.7 ± 11 days, n = 3). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21 and subjected to enrichment analysis. Variant calling was performed following GATKv3.8 best practice. The comparison of E. coli(+) EARLY and CONT cows found the up-regulation of 1090 DEGs, mainly with immune and inflammatory functions. The key signalling pathways involved NOD-like and interleukin-1 receptors and chemokines. Many up-regulated DEGs encoded antimicrobial peptides including cathelicidins, beta-defensins, S100 calcium binding proteins, haptoglobin and lactoferrin. Inflammation had largely resolved in the E. coli(+) LATE group, with only 29 up-regulated DEGs. Both EARLY and LATE cows had up-regulated DEGs encoding ATP binding cassette (ABC) transporters and haemoglobin subunits were also up-regulated in LATE cows. Twelve candidate genetic variants were identified in DEGs between the infected and CONT cows. Three were in contiguous genes WIPI1, ARSG and SLC16A6 on BTA19. Two others (RAC2 and ARHGAP26) encode a Rho-family GTPase and Rho GTPase-activating protein 26. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite prompt treatment and provide preliminary evidence for genetic differences between cows that may predispose them to infection

Associations between Circulating IGF-1 Concentrations, Disease Status and the Leukocyte Transcriptome in Early Lactation Dairy Cows

Publication history: Accepted - 19 November 2021; Published - 25 November 2021.Peripartum dairy cows commonly experience negative energy balance (EB) and immunosuppression together with high incidences of infectious and metabolic disease. This study investigated mechanisms linking EB status with immune defense in early lactation. Data were collected from multiparous Holstein cows from six herds and leukocyte transcriptomes were analyzed using RNA sequencing. Global gene expression was related to circulating IGF-1 (as a biomarker for EB) by subdividing animals into three groups, defined as IGF-1 LOW (100 ng/mL, n = 43) at 14 ± 4 days in milk (DIM). Differentially expressed genes between groups were identified using CLC Genomics Workbench V21, followed by cluster and KEGG pathway analysis, focusing on the comparison between LOW and HIGH IGF-1 cows. LOW cows were older and had significantly lower dry matter intakes and EB values, whereas HIGH cows produced more milk. During the first 35 DIM, 63% of LOW cows had more than one health problem vs. 26% HIGH cows, including more with clinical mastitis and uterine infections. Gene expression analysis indicated that leukocytes in LOW cows switched energy metabolism from oxidative phosphorylation to aerobic glycolysis (PGM, LDH, and PDK4). Many antimicrobial peptides were up-regulated in LOW cows (e.g., PTX3, DMBT1, S100A8, and S100A9) together with genes associated with inflammation, platelet activation and the complement cascade. HIGH cows had greater expression of genes regulating T and B cell function and the cytoskeleton. Overall, results suggested an ongoing cycle of poor EB and higher infection rates in LOW IGF-1 cows which was reflected in altered leukocyte functionality and reduced milk production.This project received funding from the European Union’s Seventh Framework Programme (Brussels, Belgium) for research, technological development, and demonstration under grant agreement no. 61368

Genetic Loci Involved in Antibody Response to Mycobacterium avium ssp. paratuberculosis in Cattle

Background: Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic enteritis in a wide range of animal species. In cattle, MAP causes a chronic disease called Johne's disease, or paratuberculosis, that is not treatable and the efficacy of vaccine control is controversial. The clinical phase of the disease is characterised by diarrhoea, weight loss, drop in milk production and eventually death. Susceptibility to MAP infection is heritable with heritability estimates ranging from 0.06 to 0.10. There have been several studies over the last few years that have identified genetic loci putatively associated with MAP susceptibility, however, with the availability of genome-wide high density SNP maker panels it is now possible to carry out association studies that have higher precision. Methodology/Principal Findings: The objective of the current study was to localize genes having an impact on Johne's disease susceptibility using the latest bovine genome information and a high density SNP panel (Illumina BovineSNP50 BeadChip) to perform a case/control, genome-wide association analysis. Samples from MAP case and negative controls were selected from field samples collected in 2007 and 2008 in the province of Lombardy, Italy. Cases were defined as animals serologically positive for MAP by ELISA. In total 966 samples were genotyped: 483 MAP ELISA positive and 483 ELISA negative. Samples were selected randomly among those collected from 119 farms which had at least one positive animal. Conclusion/Significance: The analysis of the genotype data identified several chromosomal regions associated with disease status: a region on chromosome 12 with high significance (P<5 710-6), while regions on chromosome 9, 11, and 12 had moderate significance (P<5 710-5). These results provide evidence for genetic loci involved in the humoral response to MAP. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information into breeding programmes for the improvement of health status

Randomized controlled trial of molnupiravir SARS-CoV-2 viral and antibody response in at-risk adult outpatients

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not 77 been elucidated in at-risk populations. Non-hospitalised participants within 5 days of 78 SARS-CoV-2 symptoms randomised to receive molnupiravir (n=253) or Usual Care 79 (n=324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated

Copy Number Variants in Two Northernmost Cattle Breeds Are Related to Their Adaptive Phenotypes

Copy number variations (CNVs) are genomic structural variants with potential functional and evolutionary effects on phenotypes. In this study, we report the identification and characterization of CNVs from the whole-genome resequencing data of two northernmost cattle breeds from Russia: the Yakut and Kholmogory cattle and their phylogenetically most related breeds, Hanwoo and Holstein, respectively. Comparisons of the CNV regions (CNVRs) among the breeds led to the identification of breed-specific CNVRs shared by cold-adapted Kholmogory and Yakut cattle. An investigation of their overlap with genes, regulatory domains, conserved non-coding elements (CNEs), enhancers, and quantitative trait loci (QTLs) was performed to further explore breed-specific biology and adaptations. We found CNVRs enriched for gene ontology terms related to adaptation to environments in both the Kholmogory and Yakut breeds and related to thermoregulation specifically in Yakut cattle. Interestingly, the latter has also been supported when exploring the enrichment of breed-specific CNVRs in the regulatory domains and enhancers, CNEs, and QTLs implying the potential contribution of CNVR to the Yakut and Kholmogory cattle breeds&rsquo; adaptation to a harsh environment

Global transcriptomic profiles of circulating leucocytes in early lactation cows with clinical or subclinical mastitis.

Bovine mastitis, an inflammatory disease of the mammary gland, is classified as subclinical or clinical. Circulating neutrophils are recruited to the udder to combat infection. We compared the transcriptomic profiles in circulating leukocytes between healthy cows and those with naturally occurring subclinical or clinical mastitis. Holstein Friesian dairy cows from six farms in EU countries were recruited. Based on milk somatic cell count and clinical records, cows were classified as healthy (n = 147), subclinically (n = 45) or clinically mastitic (n = 22). Circulating leukocyte RNA was sequenced with Illumina NextSeq single end reads (30 M). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21, followed by GO enrichment analysis. Both subclinical and clinical mastitis caused significant changes in the leukocyte transcriptome, with more intensive changes attributed to clinical mastitis. We detected 769 DEGs between clinical and healthy groups, 258 DEGs between subclinical and healthy groups and 193 DEGs between clinical and subclinical groups. Most DEGs were associated with cell killing and immune processes. Many upregulated DEGs in clinical mastitis encoded antimicrobial peptides (AZU1, BCL3, CAMP, CATHL1, CATHL2, CATHL4,CATHL5, CATHL6, CCL1, CXCL2, CXCL13, DEFB1, DEFB10, DEFB4A, DEFB7, LCN2, PGLYRP1, PRTN3, PTX3, S100A8, S100A9, S100A12, SLC11A1, TF and LTF) which were not upregulated in subclinical mastitis. The use of transcriptomic profiles has identified a much greater up-regulation of genes encoding antimicrobial peptides in circulating leukocytes of cows with naturally occurring clinical compared with subclinical mastitis. These could play a key role in combatting disease organisms