No correlative evidence of costs of infection or immunity on leucocyte telomere length in a wild population of Soay sheep.

Telomere length (TL) is a biomarker hypothesized to capture evolutionarily and ecologically important physiological costs of reproduction, infection and immunity. Few studies have estimated the relationships among infection status, immunity, TL and fitness in natural systems. The hypothesis that short telomeres predict reduced survival because they reflect costly consequences of infection and immune investment remains largely untested. Using longitudinal data from a free-living Soay sheep population, we tested whether leucocyte TL was predicted by infection with nematode parasites and antibody levels against those parasites. Helminth parasite burdens were positively associated with leucocyte TL in both lambs and adults, which is not consistent with TL reflecting infection costs. We found no association between TL and helminth-specific IgG levels in either young or old individuals which suggests TL does not reflect costs of an activated immune response or immunosenescence. Furthermore, we found no support for TL acting as a mediator of trade-offs between infection, immunity and subsequent survival in the wild. Our results suggest that while variation in TL could reflect short-term variation in resource investment or environmental conditions, it does not capture costs of infection and immunity, nor does it behave like a marker of an individual's helminth-specific antibody immune response.

Whole of government approaches to accelerate adolescent success: efficiency and financing considerations.

The multiple domains of development covered by the Sustainable Development Goals (SDGs) present a practical challenge for governments. This is particularly acute in highly resource-constrained settings which use a sector-by-sector approach to structure financing and prioritization. One potentially under-prioritized solution is to implement interventions with the potential to simultaneously improve multiple outcomes across sectors, what United Nations Development Programme refer to as development 'accelerators'. An increasing number of accelerators are being identified in the literature. There are, however, challenges associated with the evaluation and implementation of accelerators. First, as accelerators have multiple benefits, possibly in different sectors, they will be undervalued if the priority setting is conducted sector-by-sector. Second, even if their value is recognized, accelerators may not be adopted if doing so clashes with any of the multiple competing interests policymakers consider, of which efficiency/social desirability is but one. To illustrate the first challenge, and outline a possible solution, we conduct a cost-effectiveness analysis comparing the implementation of three sector-specific interventions to an accelerator, first using a sector-by-sector planning perspective, then a whole of government approach. The case study demonstrates how evaluating the cost-effectiveness of interventions sector-by-sector can lead to suboptimal efficiency rankings and overlook interventions that are efficient from a whole of government perspective. We then examine why recommendations based on a whole of government approach to evaluation are unlikely to be heeded. To overcome this second challenge, we outline a menu of existing and novel financing mechanisms that aim to address the mismatch between political incentives and logistical constraints in the priority setting and the economic evaluation evidence for cost-effective accelerators. These approaches to financing accelerators have the potential to improve efficiency, and in doing so, progress towards the SDGs, by aligning political incentives more closely with recommendations based on efficiency rankings.

Measuring the cost-effectiveness of a home-visiting intervention to promote early child development among rural families linked to the Rwandan social protection system.

Early childhood development (ECD) programmes are heralded as a way to improve children's health and educational outcomes. However, few studies in developing countries calculate the effectiveness of quality early childhood interventions. This study estimates the cost and cost-effectiveness of the Sugira Muryango (SM) trial, a home-visiting intervention to improve ECD outcomes through positive parent-child relationships. Cost-effectiveness analysis of ECD interventions is challenging given their potential to have multiple benefits. We propose a cost-effectiveness method using a single outcome, in this case the improvement in cognitive development per home-visit session, as an indication of efficiency comparable across similar interventions. The trial intervention cost US$456 per family. This cost will likely fall below US$200 if the intervention is scaled through government systems. The cost-effectiveness analysis suggests that while SM generated a relatively small impact on markers of early development, it did so efficiently. The observed improvements in cognitive development per home-visit are similar to other home-visiting interventions of longer duration. SM by focusing on the family had benefits beyond ECD, including reductions in violence against children and intermate partner violence, further analysis is needed to include these returns in the economic evaluation.

The impact of family support and organization on adolescents during school closure under Covid-19 lockdown regulations in an area of South Africa.

The Covid-19 pandemic and resultant disruptions to schooling presented significant challenges for many families. Well organised families have been shown to have a protective effect on adolescent wellbeing in periods of shock. At the onset of the Covid-19 pandemic, Asenze, a population-based cohort study, was conducting a third wave of data collection in peri-urban South Africa, examining risk and protective factors during adolescence. By March 2020, n = 272 adolescents and their caregivers (n = 241) in the cohort had been assessed when in-person data collection was halted by lockdown measures countrywide. During this cessation we undertook a brief telephonic qualitative sub-study to explore whether families enrolled in the cohort were able to cohabit cohesively and undertake distance learning during lockdown. A purposeful sample of 20 families (caregivers n = 20, adolescents n = 24) recently assessed in the Wave 3 of the main study, participated in semi-structured interviews. Quantitative data from Waves 1-3 of the main study was used to measure family function, adolescent cognitive function, and profile adolescent and caregivers. The quantitative and qualitative data were integrated to illustrate the dynamics of the participants' lives before and during lockdown. We found that families classified as well-organized before lockdown, were more likely to report co-operation during lockdown. Adolescents who were self-motivated, had access to smartphones or the internet, and were supported by both family and educators, were well-placed to continue their education without much disruption. However, few schools instituted distance learning. Of the adolescents who were not assisted- some studied on their own or with peers, but others did no schoolwork, hindered by a lack of digital connectivity, and poor service delivery. The experience of adolescence and caregivers in the Asenze Cohort during lockdown highlight the importance of family functioning for adolescent wellbeing in crisis, as well as the need for access to health, mental health, and social services, communication upgrades, and enhancements to the education system during peaceful times, to make a difference to young lives in times of crisis.

Development of a hydroxyflavone-labelled 4554W peptide probe for monitoring αS aggregation.

Parkinson's is the second most common neurodegenerative disease, with the number of individuals susceptible due to increase as a result of increasing life expectancy and a growing worldwide population. However, despite the number of individuals affected, all current treatments for PD are symptomatic-they alleviate symptoms, but do not slow disease progression. A major reason for the lack of disease-modifying treatments is that there are currently no methods to diagnose individuals during the earliest stages of the disease, nor are there any methods to monitor disease progression at a biochemical level. Herein, we have designed and evaluated a peptide-based probe to monitor αS aggregation, with a particular focus on the earliest stages of the aggregation process and the formation of oligomers. We have identified the peptide-probe K1 as being suitable for further development to be applied to number of applications including: inhibition of αS aggregation; as a probe to monitor αS aggregation, particularly at the earliest stages before Thioflavin-T is active; and a method to detect early-oligomers. With further development and in vivo validation, we anticipate this probe could be used for the early diagnosis of PD, a method to evaluate the effectiveness of potential therapeutics, and as a tool to help in the understanding of the onset and development of PD.

A mixed-model approach for estimating drivers of microbiota community composition and differential taxonomic abundance.

Next-generation sequencing (NGS) and metabarcoding approaches are increasingly applied to wild animal populations, but there is a disconnect between the widely applied generalized linear mixed model (GLMM) approaches commonly used to study phenotypic variation and the statistical toolkit from community ecology typically applied to metabarcoding data. Here, we describe the suitability of a novel GLMM-based approach for analyzing the taxon-specific sequence read counts derived from standard metabarcoding data. This approach allows decomposition of the contribution of different drivers to variation in community composition (e.g., age, season, individual) via interaction terms in the model random-effects structure. We provide guidance to implementing this approach and show how these models can identify how responsible specific taxonomic groups are for the effects attributed to different drivers. We applied this approach to two cross-sectional data sets from the Soay sheep population of St. Kilda. GLMMs showed agreement with dissimilarity-based approaches highlighting the substantial contribution of age and minimal contribution of season to microbiota community compositions, and simultaneously estimated the contribution of other technical and biological factors. We further used model predictions to show that age effects were principally due to increases in taxa of the phylum Bacteroidetes and declines in taxa of the phylum Firmicutes. This approach offers a powerful means for understanding the influence of drivers of community structure derived from metabarcoding data. We discuss how our approach could be readily adapted to allow researchers to estimate contributions of additional factors such as host or microbe phylogeny to answer emerging questions surrounding the ecological and evolutionary roles of within-host communities. IMPORTANCE NGS and fecal metabarcoding methods have provided powerful opportunities to study the wild gut microbiome. A wealth of data is, therefore, amassing across wild systems, generating the need for analytical approaches that can appropriately investigate simultaneous factors at the host and environmental scale that determine the composition of these communities. Here, we describe a generalized linear mixed-effects model (GLMM) approach to analyze read count data from metabarcoding of the gut microbiota, allowing us to quantify the contributions of multiple host and environmental factors to within-host community structure. Our approach provides outputs that are familiar to a majority of field ecologists and can be run using any standard mixed-effects modeling packages. We illustrate this approach using two metabarcoding data sets from the Soay sheep population of St. Kilda investigating age and season effects as worked examples.

Investing in human development and building state resilience in fragile contexts: A case study of early nutrition investments in Burkina Faso.

Maternal and early malnutrition have negative health and developmental impacts over the life-course. Consequently, early nutrition support can provide significant benefits into later life, provided the later life contexts allow. This study examines the limits of siloed investments in nutrition and illustrates how ignoring life-course contextual constraints limits human development benefits and exacerbates inequality, particularly in fragile contexts. This case study focuses on Burkina Faso, a country with high rates of early malnutrition and a fragile state. We modelled the impact of scaling up 10 nutrition interventions to 80% coverage for a single year cohort on stunting, nationally and sub-nationally, using the Lives Saved Tool (LiST), and the consequent impact on earnings, without and with a complementary cash-transfer in later life. The impact on earnings was modelled utilising the well-established pathway between early nutrition, years of completed schooling and, consequent adult earnings. Productivity returns were estimated as the present value of increased income over individuals' working lives, then compared to estimates of the present value of providing the cost of nutrition interventions and cash-transfers. The cost benefit ratio at the national level for scaled nutrition alone is 1:1. Sub-nationally the worst-off region yields the lowest ratio < 0.2 for every dollar spent. The combination of nutrition and cash-transfers national cost benefit is 1:12, still with regional variation but with great improvement in the poorest region. This study shows that early nutrition support alone may not be enough to address inequality and may add to state fragility. Taking a life-course perspective when priority-setting in contexts with multiple constraints on development can help to identify interventions that maximizing returns, without worsening inequality.

Anionic lipid vesicles have differential effects on the aggregation of early onset-associated α-synuclein missense mutants.

α-synuclein (αS) is the key component of synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. αS was first linked to PD through the identification of point mutations in the SNCA gene, causing single amino acid substitutions within αS and familial autosomal dominant forms of PD that profoundly accelerated disease onset by up to several decades. At least eight single-point mutations linked to familial PD (A30G/P, E46K, H50Q, G51D, and A53T/E/V) are located in proximity of the region preceding the non-ß amyloid component (preNAC) region, strongly implicating its pathogenic role in αS-mediated cytotoxicity. Furthermore, lipids are known to be important for native αS function, where they play a key role in the regulation of synaptic vesicle docking to presynaptic membranes and dopamine transmission. However, the role of lipids in the function of mutant αS is unclear. Here, we studied αS aggregation properties of WT αS and five of the most predominant single-point missense mutants associated with early onset PD in the presence of anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine lipid vesicles. Our results highlight significant differences between aggregation rates, the number of aggregates produced, and overall fibril morphologies of WT αS and the A30P, E46K, H50Q, G51D, and A53T missense mutants in the presence of lipid vesicles. These findings have important implications regarding the interplay between the lipids required for αS function and the individual point mutations known to accelerate PD and related diseases.

Library-Derived Peptide Aggregation Modulators of Parkinson's Disease Early-Onset α-Synuclein Variants.

Parkinson's Disease (PD) is characterized by the accumulation of Lewy bodies in dopaminergic neurons. The main protein component of Lewy bodies, α-synuclein (αS), is also firmly linked to PD through the identification of a number of single point mutations that are autosomal dominant for early-onset disease. Consequently, the misfolding and subsequent aggregation of αS is thought to be a key stage in the development and progression of PD. Therefore, modulating the aggregation pathway of αS is an attractive therapeutic target. Owing to the fact that all but one of the familial mutations is located in the preNAC 45-54 region of αS, we previously designed a semi-rational library using this sequence as a design scaffold. The 45-54 peptide library was screened using a protein-fragment complementation assay approach, leading to the identification of the 4554W peptide. The peptide was subsequently found to be effective in inhibiting primary nucleation of αS, the earliest stage of the aggregation pathway. Here, we build upon this previous work by screening the same 45-54 library against five of the known αS single-point mutants that are associated with early-onset PD (A30P, E46K, H50Q, G51D, and A53T). These point mutations lead to a rapid acceleration of PD pathology by altering either the rate or type of aggregates formed. All ultimately lead to earlier disease onset and were therefore used to enforce increased assay stringency during the library screening process. The ultimate aim was to identify a peptide that is effective against not only the familial αS variant from which it has been selected but that is also effective against WT αS. Screening resulted in five peptides that shared common residues at some positions, while deviating at others. All reduced aggregation of the respective target, with several also identified to be effective at reducing aggregation when incubated with other variants. In addition, our results demonstrate that a previously optimized peptide, 4554W(N6A), is highly effective against not only WT αS but also several of the single-point mutant forms and hence is a suitable baseline for further work toward a PD therapeutic.

The Asenze Cohort Study in KwaZulu-Natal, South Africa: protocol and cohort profile.

The Asenze cohort is set in South Africa, a middle-income country impacted by one of the highest global rates of people living with HIV/AIDS and high levels of socioeconomic inequality. This longitudinal population-based cohort of children and their primary caregivers assesses household and caregiver functioning, child health, social well-being, and neuro-development from childhood through adolescence. Almost 1,600 children born at the peak of the human immunodeficiency virus epidemic (2003-2005) were followed (with their primary caregivers) in 3 waves, between 2008 and 2021, at average ages of 5, 7, and 16. Wave 3 is currently underway, having assessed over 1,100 of the original wave 1 children. Wave 4 begins in 2022. The study, with a dyadic structure, uses a broad range of measures, validated in South Africa or recommended for global use, that address physical, social and neuro-development in childhood and adolescence, and the social, health, and psychological status of children's primary caregivers. The Asenze study deepens our understanding of childhood physical, cognitive, and social abilities and/or disabilities, including risk-taking behaviors, and biological, environmental, and social determinants of health. We anticipate the findings will contribute to the development of community-informed interventions to promote well-being in this South African population and elsewhere.

Associations between MHC class II variation and phenotypic traits in a free-living sheep population.

Pathogen-mediated selection (PMS) is thought to maintain the high level of allelic diversity observed in the major histocompatibility complex (MHC) class II genes. A comprehensive way to demonstrate contemporary selection is to examine associations between MHC variation and individual fitness. As individual fitness is hard to measure, many studies examine associations between MHC variation and phenotypic traits, including direct or indirect measures of adaptive immunity thought to contribute to fitness. Here, we tested associations between MHC class II variation and five phenotypic traits measured in free-living sheep captured in August: weight, strongyle faecal egg count, and plasma IgA, IgE and IgG immunoglobulin titres against the gastrointestinal nematode parasite Teladorsagia circumcincta. We found no association between MHC class II variation and weight or strongyle faecal egg count. We did, however, find associations between MHC class II variation and immunoglobulin levels which varied with isotype, age and sex. Our results suggest associations between MHC and phenotypic traits are more likely to be found for traits more closely associated with pathogen defence than integrative traits such as bodyweight and highlight the association between MHC variation and antibodies in wild populations.

The association between female reproductive performance and leukocyte telomere length in wild Soay sheep.

Telomere length (TL), typically measured across a sample of blood cells, has emerged as an exciting potential marker of physiological state and of the costs of investment in growth and reproduction within evolutionary ecology. While there is mounting evidence from studies of wild vertebrates that short TL predicts raised subsequent mortality risk, the relationship between reproductive investment and TL is less clear cut, and previous studies report both negative and positive associations. In this study, we examined the relationship between TL and different aspects of maternal reproductive performance in a free-living population of Soay sheep. We find evidence for shorter TL in females that bred, and thus paid any costs of gestation, compared to females that did not breed. However, we found no evidence for any association between TL and litter size. Furthermore, females that invested in gestation and lactation actually had longer TL than females who only invested in gestation because their offspring died shortly after birth. We used multivariate models to decompose these associations into among- and within-individual effects, and discovered that within-individual effects were driving both the negative association between TL and gestation, and the positive association between TL and lactation. This suggests that telomere dynamics may reflect recent physiologically costly investment or variation in physiological condition, depending on the aspect of reproduction being investigated. Our results highlight the physiological complexity of vertebrate reproduction, and the need to better understand how and why different aspects of physiological variation underpinning life histories impact blood cell TL.

A Downsized and Optimised Intracellular Library-Derived Peptide Prevents Alpha-Synuclein Primary Nucleation and Toxicity Without Impacting Upon Lipid Binding.

Misfolding and aggregation of alpha-synuclein (αS) within dopaminergic neurons is a key factor in the development and progression of a group of age-related neurodegenerative diseases, termed synucleinopathies, that include Parkinson's disease (PD). We previously derived a peptide inhibitor from a 209,952-member intracellular library screen by employing the preNAC region (45-54) as a design template. At least six single-point mutations firmly linked to early-onset Parkinson's disease (E46K, H50Q, G51D, A53T/E/V) are located within this region, strongly implicating a pathogenic role within αS that leads to increased cytotoxicity. A library-derived ten residue peptide, 4554W, was consequently shown to block αS aggregation at the point of primary nucleation via lipid induction, inhibiting its conversion into downstream cytotoxic species. Here we couple truncation with a full alanine scan analysis, to establish the effect upon the αS aggregation pathway relative to 4554W. This revealed the precise residues responsible for eliciting inhibitory interaction and function, as well as those potentially amenable to modification or functionalisation. We find that modification N6A combined with N-terminal truncation results in a peptide of significantly increased efficacy. Importantly, our data demonstrate that the peptide does not directly disrupt αS lipid-binding, a desirable trait since antagonists of αS aggregation and toxicity should not impede association with small synaptic neurotransmitter vesicles, and thus not disrupt dopaminergic vesicle fusion and recycling. This work paves the way toward the major aim of deriving a highly potent peptide antagonist of αS pathogenicity without impacting on native αS function.

The role of maternally transferred antibodies in maternal performance in red deer.

Maternal effects are ubiquitous. Yet, the pathways through which maternal effects occur in wild mammals remain largely unknown. We hypothesise that maternal immune transfer is a key mechanism by which mothers can affect their offspring fitness, and that individual variation in maternally derived antibodies mainly depends on a mother's characteristics and the environmental conditions she experiences. To test this, we assayed six colostrum-derived antibodies in the plasma of 1447 neonates in a wild red deer population. Neonatal antibody levels were mainly affected by maternal genes, environmental variation and costs of prior reproductive investment. We found consistent heterogeneity in maternal performance across traits, with mothers producing the heaviest calves also having calves with more antibodies. Unexpectedly, antibody levels were not associated with calf survival. We provide a unique example of how evolutionary theory on maternal effects can be used to gain insight into the causes of maternal effects in wild populations.

Heritable variation in telomere length predicts mortality in Soay sheep.

Telomere length (TL) is considered an important biomarker of whole-organism health and aging. Across humans and other vertebrates, short telomeres are associated with increased subsequent mortality risk, but the processes responsible for this correlation remain uncertain. A key unanswered question is whether TL-mortality associations arise due to positive effects of genes or early-life environment on both an individual's average lifetime TL and their longevity, or due to more immediate effects of environmental stressors on within-individual TL loss and increased mortality risk. Addressing this question requires longitudinal TL and life history data across the entire lifetimes of many individuals, which are difficult to obtain for long-lived species like humans. Using longitudinal data and samples collected over nearly two decades, as part of a long-term study of wild Soay sheep, we dissected an observed positive association between TL and subsequent survival using multivariate quantitative genetic models. We found no evidence that telomere attrition was associated with increased mortality risk, suggesting that TL is not an important marker of biological aging or exposure to environmental stress in our study system. Instead, we find that among-individual differences in average TL are associated with increased lifespan. Our analyses suggest that this correlation between an individual's average TL and lifespan has a genetic basis. This demonstrates that TL has the potential to evolve under natural conditions, and suggests an important role of genetics underlying the widespread observation that short telomeres predict mortality.

Realising the potential human development returns to investing in early and maternal nutrition: The importance of identifying and addressing constraints over the life course.

The benefits of interventions which improve early nutrition are well recognised. These benefits, however, only accrue to the extent that later life circumstances allow. Consequently, in adverse contexts many of the benefits will never be realised, particularly for the most vulnerable, exacerbating inequality. Returns to investment in early nutrition could be improved if we identified contextual factors constraining their realisation and interventions to weaken these. We estimate cost and impact of scaling 10 nutrition interventions for a cohort of South African children born in 2021. We estimate associated declines in malnutrition and mortality, and improvements in years of schooling and future earnings. To examine the role of context over the life-course we estimate benefits with and without additional improvements in school quality and employment opportunities by socio-economic quintile. Scale up reduces national stunting (height for age < = -2SD) rates among children at 24 months by 3.18 percentage points, implying an increase in mean height for age z-score (HAZ) of 0.10, and 53,000 years of additional schooling. Quintile 1 (the poorest) displays the largest decline in stunting, and largest increase in mean HAZ. Estimated total cost of increasing coverage of the interventions for the cohort is US$90 million. The present value of the additional years of schooling is estimated at close to US$2 billion. Cost-benefit ratios suggest the highest return occurs in quintile 5 (1:23). Reducing inequality in school quality closes the gap between quintile 5 and the lower quintiles. If school quality and labour force participation were equal the highest returns are in quintile 1(1:31). An enabling environment is key to maximising human development returns from investing in early nutrition, and to avoid exacerbating existing inequality. Therefore, particularly for children in adverse conditions, it is essential to identify and implement complementary interventions over the life course.

The Library Derived 4554W Peptide Inhibits Primary Nucleation of α-Synuclein.

Aggregation of α-Synuclein (αS) is widely regarded as a key factor in neuronal cell death, leading to a wide range of synucleinopathies, including Parkinson's Disease. Development of therapeutics has therefore focused on inhibiting aggregation of αS into toxic forms. One such inhibitor, based on the preNAC region αS45-54 (4554W), was identified using an intracellular peptide library screen, and subsequently shown to both inhibit formation of αS aggregates while simultaneously lowering toxicity. Subsequent efforts have sought to determine the mode of 4554W action. In particular, and consistent with the fact that both target and peptide are co-produced during library screening, we find that the peptide inhibits primary nucleation of αS, but does not modulate downstream elongation or secondary nucleation events. These findings hold significant promise towards mechanistic understanding and development of molecules that can module the first steps in αS aggregation towards novel treatments for Parkinson's disease and related synucleinopathies.

Maternally derived anti-helminth antibodies predict offspring survival in a wild mammal.

The transfer of antibodies from mother to offspring provides crucial protection against infection to offspring during early life in humans and domestic and laboratory animals. However, few studies have tested the consequences of variation in maternal antibody transfer for offspring fitness in the wild. Further, separating the immunoprotective effects of antibodies from their association with nutritional resources provided by mothers is difficult. Here, we measured plasma levels of total and parasite-specific antibodies in neonatal (less than 10 days old) wild Soay sheep over 25 years to quantify variation in maternal antibody transfer and test its association with offspring survival. Maternal antibody transfer was predicted by maternal age and previous antibody responses, and was consistent within mothers across years. Neonatal total IgG antibody levels were positively related to early growth, suggesting they reflected nutritional transfer. Neonatal parasite-specific IgG levels positively predicted first-year survival, independent of lamb weight, total IgG levels and subsequent lamb parasite-specific antibody levels. This relationship was partly mediated via an indirect negative association with parasite burden. We show that among-female variation in maternal antibody transfer can have long-term effects on offspring growth, parasite burden and fitness in the wild, and is likely to impact naturally occurring host-parasite dynamics.

Lactoferrin quantification in cattle faeces by ELISA.

BACKGROUND: Promoting and maintaining health is critical to ruminant welfare and productivity. Within human medicine, faecal lactoferrin is quantified for routine assessment of various gastrointestinal illnesses avoiding the need for blood sampling. This approach might also be adapted and applied for non-invasive health assessments in animals. METHODS: In this proof-of-concept study, a bovine lactoferrin enzyme-linked immunosorbent assays (ELISA), designed for serum and milk, was applied to a faecal supernatant to assess its potential for quantifying lactoferrin in the faeces of cattle. Faecal lactoferrin concentrations were compared to background levels to assess the viability of the technique. A comparison was then made against serum lactoferrin levels to determine if they were or were not reflective of one another. RESULTS: The optical densities of faecal samples were significantly greater than background readings, supporting the hypothesis that the assay was effective in quantifying faecal lactoferrin (T 13, 115 = 11.99, p < 0.0005). The mean faecal lactoferrin concentration was 0.269 µg mL-1 (S.E. 0.031) and the mean serum concentration 0.074 µg mL-1 (S.E. 0.005). Lactoferrin concentrations of faecal and serum samples, taken from the same animals on the same day, were significantly different (T 21 = 2.20, p = 0.039) and did not correlate (r = 0.2699, p = 0.238). CONCLUSION: Results support the hypothesis that lactoferrin can be quantified in cattle faeces by ELISA. Whilst further research is required to determine the physiological source of the lactoferrin, this highlights the potential of the method for non-invasive assessment of cattle immunology and pathology.

Prevalence and number of children living in institutional care: global, regional, and country estimates.

BACKGROUND: Children living in institutionalised settings are at risk of negative health and developmental outcomes, as well as physical and emotional abuse, yet information on their numbers is scarce. Therefore, the aim of our study was to estimate global-level, regional-level, and country-level numbers and percentages of children living in institutional care. METHODS: In this estimation study, we did a systematic review of peer-reviewed publications and a comprehensive review of surveys and unpublished literature to construct a dataset on children living in institutional care from 136 countries between 2001 and 2018. We applied a wide range of methods to estimate the number and percentages of children living in institutional care in 191 countries in 2015, the year the Sustainable Development Goals were adopted. We generated 98 sets of estimates for each dataset, with possible combinations of imputation methods for countries with different available data points. Of these 98 sets, we report here five types of global-level estimates: estimates with the highest values, those with the lowest values, those with median values, those with uncertainty levels, and those derived from methods with the smallest root-mean-square errors (RMSE). FINDINGS: Global estimates of children living in institutions in 2015 was highly sensitive to the methods and data used, ranging from 3·18 million to 9·42 million children, with a median estimate of 5·37 million. When selecting the method with the lowest RMSE, the global estimate was 4·21 million, whereas with negative binomial regression with bootstrapping, the global estimate was 7·52 (95% CI 7·48-7·56) million. We also observed large variations in country-level estimates. Compared with other regions, estimates in south Asia, sub-Saharan Africa, and Latin America had larger variations in values when switching between estimation methods. High-income countries had the highest average prevalence of institutionalisation, whereas low-income countries had the lowest average prevalence. Estimates from the full data with the smallest RMSE method showed that south Asia had the largest estimated number of children living in institutions (1·13 million), followed by Europe and central Asia (1·01 million), east Asia and Pacific (0·78 million), sub-Saharan Africa (0·65 million), Middle East and North Africa (0·30 million), Latin America and the Caribbean (0·23 million), and North America (0·09 million). North America consistently had the lowest estimates among all regions. INTERPRETATION: Worldwide, institutional care places millions of children at elevated risk of negative health and developmental outcomes, highlighting the need for deinstitutionalisation. However, there is considerable uncertainty regarding the number of children living in institutions. To improve estimates of the size of this population, we need to standardise the definition of institutional care and improve data collection, particularly in countries with large child populations. FUNDING: Lumos Foundation.