Microbiota members from body sites of dairy cows are largely shared within individual hosts throughout lactation but sharing is limited in the herd.

BACKGROUND: Host-associated microbes are major determinants of the host phenotypes. In the present study, we used dairy cows with different scores of susceptibility to mastitis with the aim to explore the relationships between microbiota composition and different factors in various body sites throughout lactation as well as the intra- and inter-animal microbial sharing. RESULTS: Microbiotas from the mouth, nose, vagina and milk of 45 lactating dairy cows were characterized by metataxonomics at four time points during the first lactation, from 1-week pre-partum to 7 months post-partum. Each site harbored a specific community that changed with time, likely reflecting physiological changes in the transition period and changes in diet and housing. Importantly, we found a significant number of microbes shared among different anatomical sites within each animal. This was between nearby anatomic sites, with up to 32% of the total number of Amplicon Sequence Variants (ASVs) of the oral microbiota shared with the nasal microbiota but also between distant ones (e.g. milk with nasal and vaginal microbiotas). In contrast, the share of microbes between animals was limited (< 7% of ASVs shared by more than 50% of the herd for a given site and time point). The latter widely shared ASVs were mainly found in the oral and nasal microbiotas. These results thus indicate that despite a common environment and diet, each animal hosted a specific set of bacteria, supporting a tight interplay between each animal and its microbiota. The score of susceptibility to mastitis was slightly but significantly related to the microbiota associated to milk suggesting a link between host genetics and microbiota. CONCLUSIONS: This work highlights an important sharing of microbes between relevant microbiotas involved in health and production at the animal level, whereas the presence of common microbes was limited between animals of the herd. This suggests a host regulation of body-associated microbiotas that seems to be differently expressed depending on the body site, as suggested by changes in the milk microbiota that were associated to genotypes of susceptibility to mastitis.

Standardized Whole Blood Assay and Bead-Based Cytokine Profiling Reveal Commonalities and Diversity of the Response to Bacteria and TLR Ligands in Cattle.

Recent developments in multiplex technologies enable the determination of a large nu\mber of soluble proteins such as cytokines in various biological samples. More than a one-by-one determination of the concentration of immune mediators, they permit the establishment of secretion profiles for a more accurate description of conditions related to infectious diseases or vaccination. Cytokine profiling has recently been made available for bovine species with the development of a Luminex® technology-based 15-plex assay. Independently from the manufacturer, we evaluated the bovine cytokine/chemokine multiplex assay for limits of detection, recovery rate, and reproducibility. Furthermore, we assessed cytokine secretion in blood samples from 107 cows upon stimulation with heat-killed bacteria and TLR2/4 ligands compared to a null condition. Secretion patterns were analyzed either using the absolute concentration of cytokines or using their relative concentration with respect to the overall secretion level induced by each stimulus. Using Partial Least Square-Discriminant Analysis, we show that the 15-cytokine profile is different under Escherichia coli, Staphylococcus aureus, and Streptococcus uberis conditions, and that IFN-γ, IL-1ß, and TNF-α contribute the most to differentiate these conditions. LPS and E. coli induced largely overlapping biological responses, but S. aureus and S. uberis were associated with distinct cytokine profiles than their respective TLR ligands. Finally, results based on adjusted or absolute cytokine levels yielded similar discriminative power, but led to different stimuli-related signatures.

Bovine Teat Cistern Microbiota Composition and Richness Are Associated With the Immune and Microbial Responses During Transition to Once-Daily Milking.

The relationship between microbiota and health has been widely reported in humans and animals. We established a link between teat cistern microbiota composition and bovine mastitis, an inflammatory disease often due to bacterial infections. To further decipher the relationships between teat cistern microbiota and immune and microbial responses, a switch from twice- to once-daily milking (ODM) in 31 initially healthy quarters of dairy cows was used to trigger an udder perturbation. In this study, a temporal relationship was reported between initial teat cistern microbiota composition and richness, the immune response to ODM, and mastitis development. Quarters with a low initial microbiota richness and taxonomic markers such as Bacteroidetes and Proteobacteria were associated with a higher rate of mastitis during ODM. Quarters with a higher richness and taxonomic markers such as Firmicutes, including the Lachnospiraceae family, and genera such as Bifidobacterium and Corynebacterium displayed early inflammation following transition to ODM but without developing mastitis (no infection). Short-term compositional shifts of microbiota indicates that microbiotas with a higher initial richness were more strongly altered by transition to ODM, with notably the disappearance of rare OTUs. Microbiota modifications were associated with an early innate immune system stimulation, which, in turn, may have contributed to the prevention of mastitis development.

Interaction between frailty and sex on mortality after elective abdominal aortic aneurysm repair.

BACKGROUND: Controversy exists surrounding gender outcome disparity and abdominal aortic aneurysm (AAA) repair. Previous reports have demonstrated worse outcomes for women undergoing open aneurysm repair (OAR); however, these differences are less evident with endovascular aneurysm repair (EVAR). Epidemiologic studies have documented that women score higher on most frailty assessment scales but paradoxically have longer life expectancy compared to men. The interaction of gender/frailty and the influence on outcomes and practice patterns surrounding EVAR and OAR is poorly described. This analysis characterizes the association of frailty/sex interactions on mortality as well as patient selection surrounding elective AAA repair in the Society for Vascular Surgery Vascular Quality Initiative. METHODS: All elective infrarenal AAA (EVAR + OAR; 2003-2017) cases were queried from the Vascular Quality Initiative database. Each patient was assigned a previously published modified frailty index (mFI) score derived from comorbidity and preoperative functional status data. Cox proportional hazard models, adjusted for statistically significant covariates, including procedural complexity, determined associations within full models and sex-stratified models. RESULTS: A total of 20,750 elective AAA cases were analyzed (EVAR 15,893 [77%]; OAR 4857 [23%]). Thirty-day mortality for EVAR and OAR was 0.7% (n = 115) and 3.5% (n = 169), respectively. Patients who died were significantly more likely to be older (EVAR, 78 vs 73 years; OAR, 74 vs 69 years; P < .0001), have larger AAA diameters (EVAR, 59 vs 56 mm; P = .005; OAR, 62 vs 59 mm; P = .001), higher mFI scores (EVAR, 3.2 vs 2.4; OAR, 3.1 vs 2.2; P < .0001), and be of female sex (EVAR hazard ratio = 1.66 [95% confidence interval, 1.10-2.52]; P = .007; OAR-1.43 [1.02-1.99]; P = .003). Significant differences in the gender distribution of frailty scores among EVAR patients were evident (mean mFI: male 2.42 vs female 2.34; P = .02), but no difference was detected for OAR (male 2.17 vs female 2.22; P = .38). The mFI was a strong independent predictor of mortality (30 days: EVAR hazard ratio = 1.36 [1.22-1.53] and OAR 1.46 [1.32-1.60]; 1 year: EVAR 1.32 [1.25-1.39] and OAR-1.38 [1.28-1.48]). There was no interaction between mFI and gender on the association with mortality. Across frailty strata, male patients were nearly twofold more likely to undergo either elective EVAR or OAR for an AAA below recommended minimum diameter thresholds (male, <5.5 cm; female, <5.0 cm). Greater mFI score did not alter OAR selection but was associated with less frequent EVAR of small AAA. CONCLUSIONS: Given the strong association between frailty and postoperative mortality, mFI can be used as a predictive tool to aid in surgical planning of patients undergoing elective AAA repair. While mFI can predict postoperative mortality for both men and women, it does not account for the survival disparity between sexes, and further research is warranted to explain this difference. There appear to be significant gender differences in patient selection based on current Society for Vascular Surgery-endorsed treatment thresholds that may have important implications on the appropriateness of AAA care delivery nationally.

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

The spectrum of hand dysfunction after hemodialysis fistula placement.

INTRODUCTION: Contemporary dogma has classically attributed hand dysfunction following hemodialysis arteriovenous fistula (AVF) placement to regional ischemia. We hypothesize that hemodynamic perturbations alone do not entirely explain the postoperative changes in hand function and, furthermore, that various elements of hand function are differentially affected following surgery. METHODS: Bilateral wrist and digital pressures and upper extremity nerve conduction tests were recorded preoperatively and at 6 weeks and 6 months following upper extremity AVF construction in 46 patients. Concurrently, biomechanical tests were administered to evaluate multiple limb functional domains including grip strength, dexterity, sensation and perception of hand function. RESULTS: Mean age was 59±14 years (75% male) and 48% were on hemodialysis at the time of access placement. 69% had a brachial-based AVF, and the remainder had radial-based accesses. Six weeks following AVF placement, a significant decrease in access side digital pressures was observed, with only partial recovery at 6 months (P<0.0001). Grip strength was significantly worse in the access side limb (P=0.0003), and Disability of Arm, Shoulder and Hand Questionnaire (DASH) score substantially worsened postoperatively (P=0.06). Digital sensation and limb dexterity did not differ between limb sides (P>0.1) or change significantly over time (P>0.1). Principal component analyses demonstrated that nerve conduction parameters tended to track the biomechanical parameters, yet both were relatively independent of the hemodynamic parameters. CONCLUSION: Our findings suggest that ischemia alone does not completely explain access-related hand dysfunction and that future study is needed to elucidate alternative mechanisms.

A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds.

BACKGROUND: Domestication and artificial selection have resulted in strong genetic drift, relaxation of purifying selection and accumulation of deleterious mutations. As a consequence, bovine breeds experience regular outbreaks of recessive genetic defects which might represent only the tip of the iceberg since their detection depends on the observation of affected animals with distinctive symptoms. Thus, recessive mutations resulting in embryonic mortality or in non-specific symptoms are likely to be missed. The increasing availability of whole-genome sequences has opened new research avenues such as reverse genetics for their investigation. Our aim was to characterize the genetic load of 15 European breeds using data from the 1000 bull genomes consortium and prove that widespread harmful mutations remain to be detected. RESULTS: We listed 2489 putative deleterious variants (in 1923 genes) segregating at a minimal frequency of 5 % in at least one of the breeds studied. Gene enrichment analysis showed major enrichment for genes related to nervous, visual and auditory systems, and moderate enrichment for genes related to cardiovascular and musculoskeletal systems. For verification purposes, we investigated the phenotypic consequences of a frameshift variant in the retinitis pigmentosa-1 gene segregating in several breeds and at a high frequency (27 %) in Normande cattle. As described in certain human patients, clinical and histological examination revealed that this mutation causes progressive degeneration of photoreceptors leading to complete blindness in homozygotes. We established that the deleterious allele was even more frequent in the Normande breed before 1975 (>40 %) and has been progressively counter-selected likely because of its associated negative effect on udder morphology. Finally, using identity-by-descent analysis we demonstrated that this mutation resulted from a unique ancestral event that dates back to ~2800 to 4000 years. CONCLUSIONS: We provide a list of mutations that likely represent a substantial part of the genetic load of domestication in European cattle. We demonstrate that they accumulated non-randomly and that genes related to cognition and sensory functions are particularly affected. Finally, we describe an ancestral deleterious variant segregating in different breeds causing progressive retinal degeneration and irreversible blindness in adult animals.

C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle.

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes.

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

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.

Anti-Müllerian hormone: a predictive marker of embryo production in cattle?

In cattle, the embryo production rate after superovulation varies between individuals and is difficult to predict. Recently, we proposed that anti-Müllerian hormone (AMH) plasma levels measured before treatment can help predict superovulatory responses. To establish whether blood measurement of AMH can help predict the number of embryos produced by a given cow after superovulation, data collected over 4 years from 45 dairy cows submitted to repeated embryo production were analysed in a retrospective study. A high within-animal repeatability (0.38 and 0.36) and a strong effect of the father of the donor cow (P < 0.01) were observed for the numbers of collected and transferable embryos, respectively. AMH concentration, measured in the plasma of donor cows during first lactation and several months before the start of the embryo production campaigns, was found to be highly correlated with the maximal number of collected (P < 0.0001) and transferable (P < 0.01) embryos per cow. In conclusion, the capacity of embryo production is a repeatable and probably heritable trait in the cow, and blood measurement of AMH in potential donor cows could be of value in determining a cow's intrinsic capacity to produce transferable embryos.