One Health security lessons from a year-long webinar series on international COVID-19 response.

Following the principles outlined by the Global Outbreak Alert and Response Network, the Federal Bureau of Investigation's International Biosecurity and Prevention Forum, the European Commission's Joint Research Centre, and the Middlebury Institute of International Studies' James Martin Center for Nonproliferation Studies cohosted a webinar series from April 2020 to January 2021 on COVID-19 management across Africa, Europe, and North America. We provide here an overview of the webinar series and discuss how lessons learned during the COVID-19 pandemic and debated during the webinars can be used to bridge One Health with biological threat-driven health security. This report can be used to inform recommendations for future One Health security approaches to strengthen global capacity and multidisciplinary cooperation.

New Detection Platform for Screening Bacteria in Liquid Samples.

The development of sensitive methods for the determination of potential bacterial contamination is of upmost importance for environmental monitoring and food safety. In this study, we present a new method combining a fast pre-enrichment step using a microporous cryogel and a detection and identification step using antimicrobial peptides (AMPs) and labelled antibodies, respectively. The experimental method consists of: (i) the capture of large amounts of bacteria from liquid samples by using a highly porous and functionalized cryogel; (ii) the detection and categorisation of Gram-positive and Gram-negative bacteria by determining their affinities toward a small set of AMPs; and (iii) the identification of the bacterial strain by using labelled detection antibodies. As proof of concept, the assessment of the three steps of the analysis was performed by using Escherichia coli and Bacillus sp. as models for Gram-negative and Gram-positive bacteria, respectively. The use of AMPs with broad specificity combined with labelled antibodies enabled the detection and potential categorization of a large spectrum of unknown or unexpected bacteria.

Initial impacts of global risk mitigation measures taken during the combatting of the COVID-19 pandemic.

This paper presents an analysis of risk mitigation measures taken by countries around the world facing the current COVID-19 outbreak. In light of the current pandemic the authors collated and clustered (using harmonised terminology) the risk mitigation measures taken around the globe in the combat to contain, and since March 11, 2020, to limit the spread of the SARS-CoV-2 virus known to cause the Coronavirus disease 2019 (COVID-19). This overview gathers lessons learnt, providing an update on the current knowledge for authorities, sectors and first responders on the effectiveness of said measures, and may allow enhanced prevention, preparedness and response for future outbreaks. Various measures such as mobility restrictions, physical distancing, hygienic measures, socio-economic restrictions, communication and international support mechanisms have been clustered and are reviewed in terms of the nature of the actions taken and their qualitative early-perceived impact. At the time of writing, it is still too premature to express the quantitative effectiveness of each risk mitigation cluster, but it seems that the best mitigation results are reported when applying a combination of voluntary and enforceable measures.

Genome wide association study of 40 clinical measurements in eight dog breeds.

The domestic dog represents an ideal model for identifying susceptibility genes, many of which are shared with humans. In this study, we investigated the genetic contribution to individual differences in 40 clinically important measurements by a genome-wide association study (GWAS) in a multinational cohort of 472 healthy dogs from eight breeds. Meta-analysis using the binary effects model after breed-specific GWAS, identified 13 genome-wide significant associations, three of them showed experimental-wide significant associations. We detected a signal at chromosome 13 for the serum concentration of alanine aminotransferase (ALT) in which we detected four breed-specific signals. A large proportion of the variance of ALT (18.1-47.7%) was explained by this locus. Similarly, a single SNP was also responsible for a large proportion of the variance (6.8-78.4%) for other measurements such as fructosamine, stress during physical exam, glucose, and morphometric measurements. The genetic contribution of single variant was much larger than in humans. These findings illustrate the importance of performing meta-analysis after breed-specific GWAS to reveal the genetic contribution to individual differences in clinically important measurements, which would lead to improvement of veterinary medicine.

Interbreed variation of biomarkers of lipid and glucose metabolism in dogs.

BACKGROUND: Markers of lipid and glucose metabolism are used in both clinical practice and research. Detection of abnormal laboratory results often relies on species-specific reference intervals, but interbreed variation can also affect data interpretation. OBJECTIVES: The purpose of the present study was to compare concentrations of selected biochemical variables among different dog breeds. METHODS: We analyzed a database containing information on biochemical variables from 534 dogs belonging to nine different breeds. All dogs were confirmed to be healthy based on history, physical examination, and ancillary tests. Concentrations of glucose, fructosamine, insulin, cholesterol, triglycerides, fatty acids, and C-reactive protein were compared using the nonparametric Kruskal-Wallis and Dunn's tests. RESULTS: All variables tested showed significant interbreed differences, although all breeds remained within the previously established RIs for dogs. Fructosamine, insulin, and cholesterol showed a wide interbreed variation that could affect the interpretation of results. CONCLUSIONS: Breed is an important factor to consider when assessing energy metabolism in dogs, especially for markers like fructosamine, insulin, and cholesterol, which vary considerably among breeds.

Influence of antral follicle size on oocyte characteristics and embryo development in the bovine.

The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter > or = 6 mm always gave a higher blastocyst rate than oocytes from follicles < 4 mm (UCL: 42% versus 14%, DIAS: 50% versus 35%, INRA: 39% versus 22%; P < 0.05). Blastocyst cell number was not affected by follicle size. Several parameters were investigated for these oocytes. The energy metabolism of cumulus-oocyte-complexes and of denuded oocytes was assessed by the oxygen and pyruvate uptake and by lactate release both at the beginning and the end of the maturation. No effect of follicle size could be detected but lactate release increased after maturation. The global profile of transcripts, the pattern of protein neosynthesis and the kinetics of meiosis resumption were not affected by follicle size. The developmental kinetics of derived embryos was also analysed. Whatever the follicle size, viable embryos had a shorter first and third embryonic cell cycle. Among the viable embryos, the size of the follicle interfered with the fourth cell cycle duration. A higher percentage of blastocysts issued from large follicle presented a short fourth cell cycle (9h) (35% versus 6%; P < 0.05). Beside, blastocysts derived from small follicles had a delayed cavitation and expansion. Thereby, a higher developmental competence for oocytes from follicle > or = 6 mm versus < 4 mm was demonstrated in three laboratories although no differences could be displayed directly at the oocyte level.

The Shepherds' Tale: A Genome-Wide Study across 9 Dog Breeds Implicates Two Loci in the Regulation of Fructosamine Serum Concentration in Belgian Shepherds.

Diabetes mellitus is a serious health problem in both dogs and humans. Certain dog breeds show high prevalence of the disease, whereas other breeds are at low risk. Fructosamine and glycated haemoglobin (HbA1c) are two major biomarkers of glycaemia, where serum concentrations reflect glucose turnover over the past few weeks to months. In this study, we searched for genetic factors influencing variation in serum fructosamine concentration in healthy dogs using data from nine dog breeds. Considering all breeds together, we did not find any genome-wide significant associations to fructosamine serum concentration. However, by performing breed-specific analyses we revealed an association on chromosome 3 (pcorrected ≈ 1:68 × 10-6) in Belgian shepherd dogs of the Malinois subtype. The associated region and its close neighbourhood harbours interesting candidate genes such as LETM1 and GAPDH that are important in glucose metabolism and have previously been implicated in the aetiology of diabetes mellitus. To further explore the genetics of this breed specificity, we screened the genome for reduced heterozygosity stretches private to the Belgian shepherd breed. This revealed a region with reduced heterozygosity that shows a statistically significant interaction (p = 0.025) with the association region on chromosome 3. This region also harbours some interesting candidate genes and regulatory regions but the exact mechanisms underlying the interaction are still unknown. Nevertheless, this finding provides a plausible explanation for breed-specific genetic effects for complex traits in dogs. Shepherd breeds are at low risk of developing diabetes mellitus. The findings in Belgian shepherds could be connected to a protective mechanism against the disease. Further insight into the regulation of glucose metabolism could improve diagnostic and therapeutic methods for diabetes mellitus.

LUPA: a European initiative taking advantage of the canine genome architecture for unravelling complex disorders in both human and dogs.

The domestic dog offers a unique opportunity to explore the genetic basis of disease, morphology and behaviour. Humans share many diseases with our canine companions, making dogs an ideal model organism for comparative disease genetics. Using newly developed resources, genome-wide association studies in dog breeds are proving to be exceptionally powerful. Towards this aim, veterinarians and geneticists from 12 European countries are collaborating to collect and analyse the DNA from large cohorts of dogs suffering from a range of carefully defined diseases of relevance to human health. This project, named LUPA, has already delivered considerable results. The consortium has collaborated to develop a new high density single nucleotide polymorphism (SNP) array. Mutations for four monogenic diseases have been identified and the information has been utilised to find mutations in human patients. Several complex diseases have been mapped and fine mapping is underway. These findings should ultimately lead to a better understanding of the molecular mechanisms underlying complex diseases in both humans and their best friend.

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs.

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

Effects of polyadenylation inhibition on meiosis progression in relation to the polyadenylation status of cyclins A2 and B1 during in vitro maturation of bovine oocytes.

The control of protein synthesis during maturation in oocytes is mainly exerted through cytoplasmic polyadenylation of stored mRNAs. We first analyzed the polyadenylation status of cyclins A2 and B1 during in vitro maturation (IVM) of bovine oocytes, using Rapid Amplification of cDNA Ends-Polyadenylation Technique (RACE-PAT). An inconstant elongation of the poly(A) tail was observed for cyclin A2 transcripts after maturation, while a constant lengthening was observed for cyclin B1, occurring during the first 12 hr of incubation. We then evaluated the effects of the polyadenylation inhibitor 3'-deoxyadenosine (3'-dA), on polyadenylation and nuclear maturation. The presence of 0.02 mM 3'-dA during the whole incubation period or from 6 hr after its beginning completely prevented meiosis progression in 100% of the oocytes. Polyadenylation of cyclin B1 was also completely prevented when 3'-dA was added at 0 hr, and greatly reduced when added at 6 hr. When 3'-dA was added at 12 hr, around metaphase I (MI), 46.9% of the oocytes have reached metaphase II (MII, vs. 78.8% in the control group) at 24 hr. The use of the same concentration of 3'-deoxyguanosine (3'-dG), that impairs transcription but not polyadenylation, did not affect cyclins polyadenylation, nor nuclear maturation, whatever was the timing of addition. These results suggest that the polyadenylation of cyclin B1 could be related to the first peak of activity of MPF, occurring around MI (10-12 hr after the onset of the maturation period). They also show that, in our culture conditions, inhibition of polyadenylation prevents meiosis progression, especially up to the MI stage, while inhibition of transcription does not.

Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos.

Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos.

Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study.

Porcine embryo selection prior to transfer is mainly influenced by morphological criteria. However, the relationship between embryonic morphology, developmental potential and cell death by apoptosis in porcine embryos is still unclear. The aim of this study was to establish embryo quality parameters for in vivo fertilised porcine embryos based on timing of development in vitro, embryo morphology and the presence of apoptosis. The kinetics of development and morphological parameters were investigated in a time-lapse cinematographic experiment. Possible links between embryo morphology and apoptosis were examined via a confocal laser scanning experiment, analysing nuclear changes, annexin V and terminal dUTP nick-end labelling. The timing of early cleavages was firmly linked to embryo developmental competence in vitro. Attainment of at least the 5-cell stage before 77 h post insemination and attainment of the morula stage before 102 h post insemination significantly increased the odds for reaching the early blastocyst stage. Overall, a negative effect of fragmentation percentage and fragmentation pattern on subsequent embryonic development was observed, but the developmental potential of embryos experiencing slight fragmentation (0-5%) was not different from embryos without fragmentation. Correlations detected between developmental arrest and fragmentation, and fragmentation and apoptosis were 0.60 and 0.87 (P < 0.05) respectively. Only a minority of the embryos arrested between the 1- and 4-cell stage displayed biochemical characteristics of apoptosis. Consequently, a significant correlation (0.57) between developmental arrest and apoptosis could only be established for embryos arrested after embryonic genome activation.

Poly(A) RNA is reduced by half during bovine oocyte maturation but increases when meiotic arrest is maintained with CDK inhibitors.

Variations in the amount of different RNA species were investigated during in vitro maturation of bovine oocytes. Total RNA content was estimated to be 2 ng before meiosis, and after meiosis resumption, no decrease was observed. Ribosomal RNA did not appear to be degraded either, whereas poly(A) RNA was reduced by half after meiosis resumption, from 53 pg to 25 pg per oocyte. Real-time polymerase chain reaction was performed on growth and differentiation factor-9 (GDF-9), on cyclin B1, and on two genes implicated in the resistance to oxidative stress, glucose-6-phosphate-dehydrogenase (G6PD) and peroxiredoxin-6 (PRDX6). When these transcripts were reverse-transcribed with hexamers, the amplification results were not different before or after in vitro maturation. But when reverse transcription was performed with oligo(dT), amplification was dramatically reduced after maturation, except for cyclin B1 mRNA, implying deadenylation without degradation of three transcripts. Although calf oocytes have a lower developmental competence, their poly(A) RNA contents were not different from that of cow oocytes, nor were they differently affected during maturation. When bovine oocytes were maintained in vitro under meiotic arrest with CDK inhibitors, their poly(A) RNA amount increased, but this rise did not change the poly(A) RNA level once maturation was achieved. The increase could not be observed under transcription inhibition and, when impeding transcription and adenylation, the poly(A) RNA decreased to a level normally observed after maturation, in spite of the maintenance of meiotic arrest. These results demonstrate the importance of adenylation and deadenylation processes during in vitro maturation of bovine oocytes.