Candidate circulating microRNA biomarkers in dogs with chronic pancreatitis.

BACKGROUND: Pancreatitis is an important cause of disease and death in dogs. Available circulating biomarkers are not sufficiently sensitive and specific for a definitive diagnosis. HYPOTHESIS: Circulating microRNAs would be differentially expressed in dogs with chronic pancreatitis and could have potential as diagnostic biomarkers. ANIMALS: Healthy controls (n = 19) and dogs with naturally occurring pancreatitis (n = 17). METHODS: A retrospective case-control study. Dogs with pancreatitis were included if they satisfied diagnostic criteria for pancreatitis as adjudicated by 3 experts. MicroRNA was extracted from stored serum samples and sequenced. Reads were mapped to mature microRNA sequences in the canine, mouse, and human genomes. Differentially expressed microRNAs were identified and the potential mechanistic relevance explored using Qiagen Ingenuity Pathway Analysis (IPA). RESULTS: Reads mapping to 196 mature microRNA sequences were detected. Eight circulating microRNAs were significantly differentially expressed in dogs with pancreatitis (≥2-fold change and false discovery rate <0.05). Four of these mapped to the canine genome (cfa-miR-221, cfa-miR-222, cfa-miR-23a, and cfa-miR-205). Three mapped to the murine genome (mmu-miR-484, mmu-miR-6240, mmu-miR-101a-3p) and 1 to the human genome (hsa-miR-1290). Expression in dogs with pancreatitis was higher for 7 microRNAs and lower for mmu-miR-101a-3p. Qiagen IPA demonstrated a number of the differently expressed microRNAs are involved in a common pancreatic inflammatory pathway. CONCLUSIONS: The significantly differentially expressed microRNAs represent promising candidates for further validation as diagnostic biomarkers for canine pancreatitis.

Using Integrated Bite Case Management to estimate the burden of rabies and evaluate surveillance in Oriental Mindoro, Philippines.

Background: Despite national elimination efforts, dog-mediated rabies remains endemic in the Philippines. Free provision of post-exposure prophylaxis (PEP) through the widespread establishment of Animal Bite Treatment Centers (ABTCs) has improved accessibility; however, the resulting upsurge in PEP demand is not sustainable, and human rabies deaths continue. Dog vaccination coverage also remains inadequate, and it is unclear whether surveillance is effective. Methods: Here, we used Integrated Bite Case Management (IBCM) to collect enhanced rabies surveillance data in Oriental Mindoro Province over a 3-year period (2020-2022). Adapting a probabilistic decision tree model, we estimated the burden of rabies, evaluated surveillance performance, and analyzed the costs and benefits of current rabies prevention and control practices in the province. Results: The incidence of bite patients receiving PEP was high in Oriental Mindoro Province (1,246/100,000 persons/year), though < 3% of presenting patients were deemed high-risk for rabies exposure (24/100,000 persons/year). Using a decision tree model, we estimated that around 73.8% of probable rabies-exposed patients sought PEP (95% Prediction Interval, PrI: 59.4%-81.1%) and that routine surveillance confirmed < 2% of circulating animal rabies cases, whereas IBCM resulted in a nearly fourfold increase in case detection. Furthermore, we estimated that an average of 560 (95% PrI 217-1,090) dogs may develop rabies annually in the province, equating to 3-5 cases per 1,000 dogs per year. On average, 20 to 43 human deaths were averted by PEP each year in Oriental Mindoro at an annual cost of $582,110 USD (i.e., $51.44 USD per person) or $20,190 USD (95% PrI $11,565-79,400) per death averted. Conclusion: While current practices for PEP provisioning in the Philippines have improved access, a large proportion of people exposed to rabies (> 26%, 95% PrI 18.8%-40.1%) are still not seeking healthcare. Integrating an intersectoral surveillance system, such as IBCM, into national policy could greatly improve case detection if well implemented, with further benefits extending to guidance for PEP administration, potentially reducing unnecessary expenditure on PEP, and situational awareness to inform control of rabies through mass dog vaccination.

Self-Adjuvanting Calcium-Phosphate-Coated Microcrystal-Based Vaccines Induce Pyroptosis in Human and Livestock Immune Cells.

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1ß and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.

Assessing the effect of a canine surgical-neutering educational programme on the knowledge and confidence of Indian veterinary participants.

India has a large, free-roaming dog population, encompassing both owned and stray dogs. Canine surgical neutering is often a central component of dog population management and rabies control initiatives. The provision of practical, surgical training opportunities remains a major challenge for veterinary educational establishments worldwide to ensure competency in this routine procedure. A 12-day educational programme, focusing on surgical neutering skills, was developed to address this need. A questionnaire comprising 26 questions covering surgical and clinical topics, and a self-assessment of confidence in undertaking five common surgical procedures, was completed immediately before and after finishing the programme. A total of 296 participants attended, with 228 achieving the inclusion criteria for the study. Total knowledge scores increased significantly after the training programme (mean score pre-18.94, 95% CI 18.13-19.74; post-28.11, 95% CI 27.44-28.77, p < 0.05) with improvements seen in all categories (surgical principles, anaesthesia, antibiotic use and wound management). After accounting for other participants' characteristics, scores increased, on average, by 9 points after training. Being female was associated with significantly higher overall scores, while compared to younger and older age groups, those aged 25-34 were associated with lower overall scores. Amongst those with post-graduate qualifications, overall scores increased with age. Furthermore, there was an increase in self-rated confidence by participants in undertaking all five procedures. This study demonstrates that a targeted training programme can improve veterinary participants' knowledge and confidence in canine surgical neutering and may provide an effective way to develop surgical expertise amongst veterinarians engaged in dog population management initiatives.

A genome-wide association study for loin depth and muscle pH in pigs from intensely selected purebred lines.

BACKGROUND: Genome-wide association studies (GWAS) aim at identifying genomic regions involved in phenotype expression, but identifying causative variants is difficult. Pig Combined Annotation Dependent Depletion (pCADD) scores provide a measure of the predicted consequences of genetic variants. Incorporating pCADD into the GWAS pipeline may help their identification. Our objective was to identify genomic regions associated with loin depth and muscle pH, and identify regions of interest for fine-mapping and further experimental work. Genotypes for ~ 40,000 single nucleotide morphisms (SNPs) were used to perform GWAS for these two traits, using de-regressed breeding values (dEBV) for 329,964 pigs from four commercial lines. Imputed sequence data was used to identify SNPs in strong ([Formula: see text] 0.80) linkage disequilibrium with lead GWAS SNPs with the highest pCADD scores. RESULTS: Fifteen distinct regions were associated with loin depth and one with loin pH at genome-wide significance. Regions on chromosomes 1, 2, 5, 7, and 16, explained between 0.06 and 3.55% of the additive genetic variance and were strongly associated with loin depth. Only a small part of the additive genetic variance in muscle pH was attributed to SNPs. The results of our pCADD analysis suggests that high-scoring pCADD variants are enriched for missense mutations. Two close but distinct regions on SSC1 were associated with loin depth, and pCADD identified the previously identified missense variant within the MC4R gene for one of the lines. For loin pH, pCADD identified a synonymous variant in the RNF25 gene (SSC15) as the most likely candidate for the muscle pH association. The missense mutation in the PRKAG3 gene known to affect glycogen content was not prioritised by pCADD for loin pH. CONCLUSIONS: For loin depth, we identified several strong candidate regions for further statistical fine-mapping that are supported in the literature, and two novel regions. For loin muscle pH, we identified one previously identified associated region. We found mixed evidence for the utility of pCADD as an extension of heuristic fine-mapping. The next step is to perform more sophisticated fine-mapping and expression quantitative trait loci (eQTL) analysis, and then interrogate candidate variants in vitro by perturbation-CRISPR assays.

Use of multi-color flow cytometry for canine immune cell characterization in cancer.

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.

The effect of season, management and endocrinopathies on vitamin D status in horses.

BACKGROUND: Vitamin D deficiency is common in humans and is increasingly linked to the pathogenesis of a multitude of diseases including obesity and metabolic syndrome. The biology of vitamin D in horses is poorly described; the relative contribution of the diet and skin synthesis to circulating concentrations is unclear and associations with the endocrine disease have not been explored. OBJECTIVES: To determine the relationship between management, season and endocrine disease and vitamin D status in horses. STUDY DESIGN: Cross-sectional cohort study. METHODS: Plasma concentrations of 25-hydroxyvitamin D2 (25(OH)D2 ) and D3 (25(OH)D3 ) were measured by liquid chromatography-tandem mass spectrometry in 34 healthy unsupplemented grazing ponies and 22 stabled Thoroughbreds receiving supplementary vitamin D3 in feed. A nested group of 18 grazing ponies were sampled on long and short days (>12 and <12 h of light/day) to determine the effect of sunlight exposure. In addition, the relationships between age, sex, adiposity, serum insulin, adrenocorticotropic hormone and vitamin D status were assessed in a mixed group of 107 horses using a linear regression model. RESULTS: All animals had a measurable level of 25(OH)D2 (median 10.7 nmol/L) whilst 25(OH)D3 was only detected in Thoroughbreds receiving D3 supplementation. Thoroughbreds had lower concentrations of 25(OH)D2 than ponies (7.4 vs. 12.6 nmol/L, p < 0.01). In grazing ponies, 25(OH)D2 concentrations were significantly higher on long days compared to short days (14.4 vs. 8.7 nmol/L, p < 0.01), whilst 25(OH)D3 was undetectable. Measures of increased adiposity, but not basal insulin, were associated with higher 25(OH)D2 concentrations, conversely to humans. Increasing ACTH was associated with lower 25(OH)D2 (p < 0.01). MAIN LIMITATIONS: Vitamin D2 concentrations were not measured in grass or forage. CONCLUSIONS: In horses 25(OH)D2 is the predominant vitamin D metabolite, and there is an apparent lack of endogenous vitamin D3 production. The relationship between vitamin D and endocrine disorders in horses does not reflect that of other species and warrants further investigation.

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation.

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species. However, the temporal changes in maternal vitamin D status throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated. The abundance of 25(OH)D in maternal plasma was quantified by HPLC-MS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (n = 5-11 gilts/GD). Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (P < 0.05). The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight, ovulation rate, mean litter weight, number of live foetuses, percentage prenatal survival, and sex ratio of the litter was assessed. Maternal 25(OH)D (P = 0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (P < 0.05) concentrations were inversely associated with gilt weight. Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90 (P < 0.05). This study has provided novel insights into temporal changes in maternal vitamin D status throughout gestation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight, percentage prenatal survival and percentage of male foetuses in the litter. Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.

Serum metabolomic profiles in dogs with chronic enteropathy.

BACKGROUND: Metabolic profiles differ between healthy humans and those with inflammatory bowel disease. Few studies have examined metabolic profiles in dogs with chronic enteropathy (CE). HYPOTHESIS: Serum metabolic profiles of dogs with CE are significantly different from those of healthy dogs. ANIMALS: Fifty-five dogs with CE and 204 healthy controls. METHODS: A cross-sectional study. The serum concentrations of 99 metabolites measured using a canine-specific proton nuclear magnetic resonance spectroscopy platform were studied. A 2-sample unpaired t-test was used to compare the 2 study samples. The threshold for significance was set at P < .05 with a Bonferroni correction for each metabolite group. RESULTS: Nineteen metabolites and 18 indices of lipoprotein composition were significantly different between the CE and healthy dogs. Four metabolites were significantly higher in dogs with CE, including phenylalanine (mean and SD) (healthy: 0.0417 mmol/L; [SD] 0.0100; CE: 0.0480 mmol/L; SD: 0.0125; P value: <.001) and lactate (healthy: 1.8751 mmol/L; SD: 0.7808; CE: 2.4827 mmol/L; SD CE: 1.4166; P value: .003). Fifteen metabolites were significantly lower in dogs with CE, including total fatty acids, and glycine (healthy: 0.2273 mmol/L; SD: 0.0794; CE: 0.1828 mmol/L; SD CE: 0.0517; P value: <.001). CONCLUSIONS AND CLINICAL IMPORTANCE: The metabolic profile of dogs with CE is significantly different from that of healthy dogs, this opens novel research avenues to develop better diagnostic and prognostic approaches as well as therapeutic trials.

Investigation of a relationship between serum concentrations of microRNA-122 and alanine aminotransferase activity in hospitalised cats.

OBJECTIVES: Current blood tests to diagnose feline liver diseases are suboptimal. Serum concentrations of microRNA (miR)-122 have been shown in humans, dogs and rodents to be a sensitive and specific biomarker for liver injury. To explore the potential diagnostic utility of measuring serum concentrations of miR-122 in cats, miR-122 was measured in a cohort of ill, hospitalised cats with known serum alanine aminotransferase (ALT) activity. METHODS: In this retrospective study, cats were grouped into those with an ALT activity within the reference interval (0-83 U/l; n = 38) and those with an abnormal ALT activity (>84 U/l; n = 25). Serum concentrations of miR-122 were measured by real-time quantitative PCR and the relationship between miR-122 and ALT was examined. RESULTS: miR-122 was significantly higher in the group with high ALT activity than the ALT group, within normal reference limits (P <0.0004). There was also a moderately positive correlation between serum ALT activity and miR-122 concentrations (P <0.001; r = 0.52). CONCLUSIONS AND RELEVANCE: Concentrations of miR-122 were reliably quantified in feline serum and were higher in a cohort of cats with increased ALT activity than in cats with normal ALT activity. This work highlights the potential diagnostic utility of miR-122 as a biomarker of liver damage in cats and encourages further investigation to determine the sensitivity and specificity of miR-122 as a biomarker of hepatocellular injury in this species.

Ranging patterns and factors associated with movement in free-roaming domestic dogs in urban Malawi.

Rabies is a neglected zoonotic disease that causes around 59,000 deaths per year globally. In Africa, rabies virus is mostly maintained in populations of free-roaming domestic dogs (FRDD) that are predominantly owned. Characterizing the roaming behavior of FRDD can provide relevant information to understand disease spread and inform prevention and control interventions. To estimate the home range (HR) of FRDD and identify predictors of HR size, we studied 168 dogs in seven different areas of Blantyre city, Malawi, tracking them with GPS collars for 1-4 days. The median core HR (HR50) of FRDD in Blantyre city was 0.2 ha (range: 0.08-3.95), while the median extended HR (HR95) was 2.14 ha (range: 0.52-23.19). Multivariable linear regression models were built to identify predictors of HR size. Males presented larger HR95 than females. Dogs living in houses with a higher number of adults had smaller HR95, while those living in houses with higher number of children had larger HR95. Animals that received products of animal origin in their diets had larger HR95, and only in the case of females, animals living in low-income areas had larger HR50 and HR95. In contrast, whether male dogs were castrated or not was not found to be associated with HR size. The results of this study may help inform rabies control and prevention interventions in Blantyre city, such as designing risk-based surveillance activities or rabies vaccination campaigns targeting certain FRDD subpopulations. Our findings can also be used in rabies awareness campaigns, particularly to illustrate the close relationship between children and their dogs.

Seasonal variation in serum metabolites of northern European dogs.

BACKGROUND: Metabolic profiling identifies seasonal variance of serum metabolites in humans. Despite the presence of seasonal disease patterns, no studies have assessed whether serum metabolites vary seasonally in dogs. HYPOTHESIS: There is seasonal variation in the serum metabolite profiles of healthy dogs. ANIMALS: Eighteen healthy, client-owned dogs. METHODS: A prospective cohort study. Serum metabolomic profiles were assessed monthly in 18 healthy dogs over a 12-month period. Metabolic profiling was conducted using a canine-specific proton nuclear magnetic resonance spectroscopy platform, and the effects of seasonality were studied for 98 metabolites using a cosinor model. Seasonal component was calculated, which describes the seasonal variation of each metabolite. RESULTS: We found no evidence of seasonal variation in 93 of 98 metabolites. Six metabolites had statistically significant seasonal variance, including cholesterol (mean 249 mg/dL [6.47 mmol/L] with a seasonal component amplitude of 9 mg/dL [0.23 mmol/L]; 95% confidence interval [CI] 6-13 mg/dL [0.14-0.33 mmol/L], P < .008), with a peak concentration of 264 mg/dL (6.83 mmol/L) in June and trough concentration of 236 mg/dL (6.12 mmol/L) in December. In contrast, there was a significantly lower concentration of lactate (mean 20 mg/dL [2.27 mmol/L] with a seasonal component amplitude of 4 mg/dL [0.42 mmol/L]; 95% CI 2-6 mg/dL [0.22-0.62 mmol/L], P < .001) during the summer months compared to the winter months, with a peak concentration of 26 mg/dL (2.9 mmol/L) in February and trough concentration of 14 mg/dL (1.57 mmol/L) in July. CONCLUSIONS AND CLINICAL IMPORTANCE: We found no clear evidence that seasonal reference ranges need to be established for serum metabolites of dogs.

Vitamin D status is heritable and under environment-dependent selection in the wild.

Vitamin D has a well-established role in skeletal health and is increasingly linked to chronic disease and mortality in humans and companion animals. Despite the clear significance of vitamin D for health and obvious implications for fitness under natural conditions, no longitudinal study has tested whether the circulating concentration of vitamin D is under natural selection in the wild. Here, we show that concentrations of dietary-derived vitamin D2 and endogenously produced vitamin D3  metabolites are heritable and largely polygenic in a wild population of Soay sheep (Ovis aries). Vitamin D2  status was positively associated with female adult survival, and vitamin D3  status predicted female fecundity in particular, good environment years when sheep density and competition for resources was low. Our study provides evidence that vitamin D status has the potential to respond to selection, and also provides new insights into how vitamin D metabolism is associated with fitness in the wild.

Implementing a One Health Approach to Rabies Surveillance: Lessons From Integrated Bite Case Management.

As part of the 'Zero by 30' strategy to end human deaths from dog-mediated rabies by 2030, international organizations recommend a One Health framework that includes Integrated Bite Case Management (IBCM). However, little is understood about the implementation of IBCM in practice. This study aims to understand how IBCM is conceptualized, exploring how IBCM has been operationalized in different contexts, as well as barriers and facilitators to implementation. Semi-structured interviews were conducted with seventeen practitioners and researchers with international, national, and local expertise across Africa, Asia, and the Americas. Thematic analysis was undertaken using both inductive and deductive approaches. Four main themes were identified: 1) stakeholders' and practitioners' conceptualization of IBCM and its role in rabies elimination; 2) variation in how IBCM operates across different contexts; 3) barriers and facilitators of IBCM implementation in relation to risk assessment, PEP provisioning, animal investigation, One Health collaboration, and data reporting; and 4) the impact of the COVID-19 pandemic on IBCM programs. This study highlights the diversity within experts' conceptualization of IBCM, and its operationalization. The range of perspectives revealed that there are different ways of organizing IBCM within health systems and it is not a one-size-fits-all approach. The issue of sustainability remains the greatest challenge to implementation. Contextual features of each location influenced the delivery and the potential impact of IBCM. Programs spanned from highly endemic settings with limited access to PEP charged to the patient, to low endemicity settings with a large patient load associated with free PEP policies and sensitization. In practice, IBCM was tailored to meet the demands of the local context and level of rabies control. Thus, experts' experiences did not necessarily translate across contexts, affecting perceptions about the function, motivation for, and implementation of IBCM. To design and implement future and current programs, guidance should be provided for health workers receiving patients on assessing the history and signs of rabies in the biting animal. The study findings provide insights in relation to implementation of IBCM and how it can support programs aiming to reach the Zero by 30 goal.

More Than a Moggy; A Population Genetics Analysis of the United Kingdom's Non-Pedigree Cats.

The domestic cat is one of the most popular pets in the world. It is estimated that 89-92% of domestic cats in the UK are non-pedigree Domestic shorthair (DSH), Domestic longhair (DLH), or Domestic semi-longhair cats (DSLH). Despite their popularity, little is known of the UK non-pedigree cats' population structure and breeding dynamics. Using a custom designed single nucleotide variant (SNV) array, this study investigated the population genetics of 1344 UK cats. Principal components analysis (PCA) and fastSTRUCTURE analysis verified that the UK's DSH, DLH, and DSLH cats are random-bred, rather than admixed, mix breed, or crossbred. In contrast to pedigree cats, the linkage disequilibrium of these random-bred cats was least extensive and decayed rapidly. Homozygosity by descent (HBD) analysis showed the majority of non-pedigree cats had proportionally less of their genome in HBD segments compared to pedigree cats, and that these segments were older. Together, these findings suggest that the DSH, DLH, and DSLH cats should be considered as a population of random-bred cats rather than a crossbred or pedigree-admixed cat. Unexpectedly, 19% of random-bred cat genomes displayed a higher proportion of HBD segments associated with more recent inbreeding events. Therefore, while non-pedigree cats as a whole are genetically diverse, they are not impervious to inbreeding and its health risks.

Vitamin D insufficiency in COVID-19 and influenza A, and critical illness survivors: a cross-sectional study.

OBJECTIVES: The steroid hormone vitamin D has roles in immunomodulation and bone health. Insufficiency is associated with susceptibility to respiratory infections. We report 25-hydroxy vitamin D (25(OH)D) measurements in hospitalised people with COVID-19 and influenza A and in survivors of critical illness to test the hypotheses that vitamin D insufficiency scales with illness severity and persists in survivors. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Plasma was obtained from 295 hospitalised people with COVID-19 (International Severe Acute Respiratory and emerging Infections Consortium (ISARIC)/WHO Clinical Characterization Protocol for Severe Emerging Infections UK study), 93 with influenza A (Mechanisms of Severe Acute Influenza Consortium (MOSAIC) study, during the 2009-2010 H1N1 pandemic) and 139 survivors of non-selected critical illness (prior to the COVID-19 pandemic). Total 25(OH)D was measured by liquid chromatography-tandem mass spectrometry. Free 25(OH)D was measured by ELISA in COVID-19 samples. OUTCOME MEASURES: Receipt of invasive mechanical ventilation (IMV) and in-hospital mortality. RESULTS: Vitamin D insufficiency (total 25(OH)D 25-50 nmol/L) and deficiency (<25 nmol/L) were prevalent in COVID-19 (29.3% and 44.4%, respectively), influenza A (47.3% and 37.6%) and critical illness survivors (30.2% and 56.8%). In COVID-19 and influenza A, total 25(OH)D measured early in illness was lower in patients who received IMV (19.6 vs 31.9 nmol/L (p<0.0001) and 22.9 vs 31.1 nmol/L (p=0.0009), respectively). In COVID-19, biologically active free 25(OH)D correlated with total 25(OH)D and was lower in patients who received IMV, but was not associated with selected circulating inflammatory mediators. CONCLUSIONS: Vitamin D deficiency/insufficiency was present in majority of hospitalised patients with COVID-19 or influenza A and correlated with severity and persisted in critical illness survivors at concentrations expected to disrupt bone metabolism. These findings support early supplementation trials to determine if insufficiency is causal in progression to severe disease, and investigation of longer-term bone health outcomes.

A genome-wide association analysis for body weight at 35 days measured on 137,343 broiler chickens.

BACKGROUND: Body weight (BW) is an economically important trait in the broiler (meat-type chickens) industry. Under the assumption of polygenicity, a "large" number of genes with "small" effects is expected to control BW. To detect such effects, a large sample size is required in genome-wide association studies (GWAS). Our objective was to conduct a GWAS for BW measured at 35 days of age with a large sample size. METHODS: The GWAS included 137,343 broilers spanning 15 pedigree generations and 392,295 imputed single nucleotide polymorphisms (SNPs). A false discovery rate of 1% was adopted to account for multiple testing when declaring significant SNPs. A Bayesian ridge regression model was implemented, using AlphaBayes, to estimate the contribution to the total genetic variance of each region harbouring significant SNPs (1 Mb up/downstream) and the combined regions harbouring non-significant SNPs. RESULTS: GWAS revealed 25 genomic regions harbouring 96 significant SNPs on 13 Gallus gallus autosomes (GGA1 to 4, 8, 10 to 15, 19 and 27), with the strongest associations on GGA4 at 65.67-66.31 Mb (Galgal4 assembly). The association of these regions points to several strong candidate genes including: (i) growth factors (GGA1, 4, 8, 13 and 14); (ii) leptin receptor overlapping transcript (LEPROT)/leptin receptor (LEPR) locus (GGA8), and the STAT3/STAT5B locus (GGA27), in connection with the JAK/STAT signalling pathway; (iii) T-box gene (TBX3/TBX5) on GGA15 and CHST11 (GGA1), which are both related to heart/skeleton development); and (iv) PLAG1 (GGA2). Combined together, these 25 genomic regions explained ~ 30% of the total genetic variance. The region harbouring significant SNPs that explained the largest portion of the total genetic variance (4.37%) was on GGA4 (~ 65.67-66.31 Mb). CONCLUSIONS: To the best of our knowledge, this is the largest GWAS that has been conducted for BW in chicken to date. In spite of the identified regions, which showed a strong association with BW, the high proportion of genetic variance attributed to regions harbouring non-significant SNPs supports the hypothesis that the genetic architecture of BW35 is polygenic and complex. Our results also suggest that a large sample size will be required for future GWAS of BW35.

Genetic architecture and major genes for backfat thickness in pig lines of diverse genetic backgrounds.

BACKGROUND: Backfat thickness is an important carcass composition trait for pork production and is commonly included in swine breeding programmes. In this paper, we report the results of a large genome-wide association study for backfat thickness using data from eight lines of diverse genetic backgrounds. METHODS: Data comprised 275,590 pigs from eight lines with diverse genetic backgrounds (breeds included Large White, Landrace, Pietrain, Hampshire, Duroc, and synthetic lines) genotyped and imputed for 71,324 single-nucleotide polymorphisms (SNPs). For each line, we estimated SNP associations using a univariate linear mixed model that accounted for genomic relationships. SNPs with significant associations were identified using a threshold of p < 10-6 and used to define genomic regions of interest. The proportion of genetic variance explained by a genomic region was estimated using a ridge regression model. RESULTS: We found significant associations with backfat thickness for 264 SNPs across 27 genomic regions. Six genomic regions were detected in three or more lines. The average estimate of the SNP-based heritability was 0.48, with estimates by line ranging from 0.30 to 0.58. The genomic regions jointly explained from 3.2 to 19.5% of the additive genetic variance of backfat thickness within a line. Individual genomic regions explained up to 8.0% of the additive genetic variance of backfat thickness within a line. Some of these 27 genomic regions also explained up to 1.6% of the additive genetic variance in lines for which the genomic region was not statistically significant. We identified 64 candidate genes with annotated functions that can be related to fat metabolism, including well-studied genes such as MC4R, IGF2, and LEPR, and more novel candidate genes such as DHCR7, FGF23, MEDAG, DGKI, and PTN. CONCLUSIONS: Our results confirm the polygenic architecture of backfat thickness and the role of genes involved in energy homeostasis, adipogenesis, fatty acid metabolism, and insulin signalling pathways for fat deposition in pigs. The results also suggest that several less well-understood metabolic pathways contribute to backfat development, such as those of phosphate, calcium, and vitamin D homeostasis.

Computed Tomography Osteodensitometry for Assessment of Bone Mineral Density of the Canine Head-Preliminary Results.

Despite bone mineral density (BMD) being regularly measured in human patients, BMD studies in clinical cohorts of dogs is lacking. In order to facilitate BMD assessment and in turn better identify dogs suffering from metabolic bone disease, rapid, easy and precise computed tomography (qCT) techniques are required. In this study we aimed to assess the utility of quantitative computed tomography (qCT) bone mineral density (BMD) measurement of the canine calvarium using a semiautomated osteodensitometry software and define host factors associated with canine bone mineral density in a skeletally healthy population. Calvarial qCT at the level of the temporomandibular joints was performed on 323 dogs using a dedicated osteodensitometry calibration phantom during a clinically indicated head computed tomography (CT). Calvarial BMD was analyzed using a dedicated semiautomatic osteodensitometry software for contouring of the calvarial lamellar bone margins and BMD calculation. The mean duration of the calvarial qCT scanning was 64.6 s, and the mean duration of BMD analysis was 34 s, with a mean of two manual adjustments required for the bone margin tracing. The median BMD of all dogs in our study was 659 mg Calcium hydroxyapatite/mL. There was a negative linear correlation between BMD and body weight, but no correlation with age, sex or neutered status. Canine BMD assessment using qCT of the calvarium is a practical and fast technique that can be added to a clinical CT examination with minimal extra time requirements. Canine BMD host-dependent factors exhibit different relationships from that of humans; however, further investigation is warranted.