Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants.

Welfare of ducks, geese and quail on farm.

This Scientific Opinion concerns the welfare of Domestic ducks (Anas platyrhynchos domesticus), Muscovy ducks (Cairina moschata domesticus) and their hybrids (Mule ducks), Domestic geese (Anser anser f. domesticus) and Japanese quail (Coturnix japonica) in relation to the rearing of breeders, birds for meat, Muscovy and Mule ducks and Domestic geese for foie gras and layer Japanese quail for egg production. The most common husbandry systems (HSs) in the European Union are described for each animal species and category. The following welfare consequences are described and assessed for each species: restriction of movement, injuries (bone lesions including fractures and dislocations, soft tissue lesions and integument damage and locomotory disorders including lameness), group stress, inability to perform comfort behaviour, inability to perform exploratory or foraging behaviour and inability to express maternal behaviour (related to prelaying and nesting behaviours). Animal-based measures relevant for the assessment of these welfare consequences were identified and described. The relevant hazards leading to the welfare consequences in the different HSs were identified. Specific factors such as space allowance (including minimum enclosure area and height) per bird, group size, floor quality, characteristics of nesting facilities and enrichment provided (including access to water to fulfil biological needs) were assessed in relation to the welfare consequences and, recommendations on how to prevent the welfare consequences were provided in a quantitative or qualitative way.

Evaluating remote post-mortem veterinary meat inspections on pig carcasses using pre-recorded video material.

BACKGROUND: Official meat inspections at small-scale slaughterhouses and game-handling establishments in geographically remote areas place a heavy burden on the meat-producing food business operators. By performing meat inspections remotely using live-streamed video, instead of on-site, the official control could meet the goals of sustainability, resilience and logistics. We investigated the agreement between the two approaches at pig slaughter. Two official veterinarians (OVs) inspected 400 pig carcasses at a Swedish slaughterhouse, with each pig being inspected on site by one OV and remotely by the other. After a period of 3 to 6 months, video recordings of the remote inspections were assessed again by the same OVs, thus enabling direct comparisons of previous on-site inspections and renewed video-based inspections within the same OV. RESULTS: Agreement across 22 finding codes was generally very high for both OVs. In all but one case (whether to fully condemn a carcass), for both OVs, Prevalence-Adjusted Bias-Adjusted kappa was well above 0.8, indicating 'almost perfect agreement'. CONCLUSIONS: This study supports earlier findings that reliable post-mortem inspections can be performed using video, and indicates higher agreement between remote and on-site inspections if the same OV performs both.

Incidence, risk factors and pre-emptive screening for COVID-19 associated pulmonary aspergillosis in an era of immunomodulant therapy.

PURPOSE: COVID-19 associated pulmonary aspergillosis (CAPA) is associated with increased morbidity and mortality in ICU patients. We investigated the incidence of, risk factors for and potential benefit of a pre-emptive screening strategy for CAPA in ICUs in the Netherlands/Belgium during immunosuppressive COVID-19 treatment. MATERIALS AND METHODS: A retrospective, observational, multicentre study was performed from September 2020-April 2021 including patients admitted to the ICU who had undergone diagnostics for CAPA. Patients were classified based on 2020 ECMM/ISHAM consensus criteria. RESULTS: CAPA was diagnosed in 295/1977 (14.9%) patients. Corticosteroids were administered to 97.1% of patients and interleukin-6 inhibitors (anti-IL-6) to 23.5%. EORTC/MSGERC host factors or treatment with anti-IL-6 with or without corticosteroids were not risk factors for CAPA. Ninety-day mortality was 65.3% (145/222) in patients with CAPA compared to 53.7% (176/328) without CAPA (p = 0.008). Median time from ICU admission to CAPA diagnosis was 12 days. Pre-emptive screening for CAPA was not associated with earlier diagnosis or reduced mortality compared to a reactive diagnostic strategy. CONCLUSIONS: CAPA is an indicator of a protracted course of a COVID-19 infection. No benefit of pre-emptive screening was observed, but prospective studies comparing pre-defined strategies would be required to confirm this observation.

Limited Effects of Pain Control Treatments on Behaviour and Weight Gain of Pure and Crossbred Nellore Heifer Calves When Subjected to Hot-Iron Branding.

Hot-iron branding is still commonly performed in cattle farming in tropical countries, and possibly has negative consequences for animal welfare and weight gain. This study examined the behavioural and weight gain responses of pure and crossbred Nellore heifer calves subjected to hot-iron branding on the cheek, without and with use of anaesthesia and analgesia. Ninety-two heifer calves, around 120 days old, were studied prospectively when subjected to hot-iron branding on the cheek (a statutory procedure in Brazil following brucellosis vaccination). Four randomly selected groups of calves were allocated to four treatments: no pain control (CO); subcutaneous anaesthetic local block (LA); intramuscular analgesia (meloxicam) (LT); and local anaesthesia plus meloxicam (LL). Behaviour, flight speed and body weight were evaluated before, during, and five (5-d) and 60 days (60-d) after branding. For these parameters, the only difference observed was higher tension in the CO group 5-d post-branding, suggesting a short-term negative effect of branding without pain control. The limited effects of the pain control treatments suggest interference in pain assessment by other factors, such as expression of fear and stress. Despite the lack of differences observed in behaviour and production parameters, facial hot-iron branding is an obvious welfare issue and, due to the additional handling involved, adoption of a simple pain relief protocol is not sufficient to minimise the welfare impact.

Brief report: community-acquired Friedlander's pneumonia and pulmonary metastatic Klebsiella pneumoniae infection caused by hypervirulent ST23 in the Netherlands.

Infections with hypervirulent Klebsiella pneumoniae (hvKp) commonly presents with primary liver infection, bacteremia, and metastatic abscesses. Here, we present 2 cases of severe community-acquired pulmonary infections by hvKp in patients in the Netherlands without recent travel history. Both bacterial isolates are closely related to an archetype ST23 hvKp reference isolate. Based on these findings, surveillance programs on hvKp may consider to include isolates from community-acquired pneumonia by K. pneumoniae.

Swedish Trotting Horse Trainers' Perceptions of Animal Welfare Inspections from Public and Private Actors.

In Sweden, the County Administrative Board (CAB) and Swedish Trotting Association (STA) both perform animal welfare inspections of the premises of trotting horse trainers. The CAB inspection checks for compliance with the legislation, and the STA inspection checks for compliance with the private 'Trotter Health Standard', which mainly sets the same requirements as the legislation. This study investigated the views of trainers on these inspections both as separate events and in relation to each other. A digital questionnaire was sent out to trotting horse trainers in Sweden during spring 2021, and 396 trainers responded. Descriptive and statistical analyses were used to evaluate the responses. In general, the trainers reported positive experiences of both the CAB and STA inspections, but they had consistently more positive views about the private STA inspections than the official CAB inspections. The outcome of the inspections, i.e., non-compliance or not, did not affect trainers' perceptions of the inspections, but inspectors' knowledge, manner, and responsiveness had a strong effect. The trainers were generally satisfied with the current control system but would like better coordination between the different inspections.

Animal Welfare and Food Safety When Slaughtering Cattle Using the Gunshot Method.

Transporting cattle from farm to slaughterhouse is often stressful for the animal, which can impair the meat quality. With the gunshot method, the animal is stunned with a rifle shot while together with familiar herd members in their home environment, exsanguinated and transported to a nearby slaughterhouse. Aiming to assess the consequences for animal welfare and food safety, 20 Hereford steers aged 18-54 months were shot with .22 Magnum ammunition from an elevated position and distance of 6-12 m. Each time, only one out of four to seven animals in a 16 × 10 m corral was shot. Dressing was done on farm. Based on the animals' behaviour and blood concentrations of cortisol, glucose and lactate, stress levels before shooting were low. Eleven animals were deeply stunned, the consciousness of seven others was ambiguous, and two were poorly stunned. Two animals were reshot. The bleed-out was satisfactory for all animals, and little or no faecal contamination was found on the carcasses. We conclude that the gunshot method is applicable to large beef steers while maintaining a satisfactory level of animal welfare and food safety, provided that the necessary conditions can be attained.

Risk Factors for Intra-Abdominal Candidiasis in Intensive Care Units: Results from EUCANDICU Study.

INTRODUCTION: Intra-abdominal infections represent the second most frequently acquired infection in the intensive care unit (ICU), with mortality rates ranging from 20% to 50%. Candida spp. may be responsible for up to 10-30% of cases. This study assesses risk factors for development of intra-abdominal candidiasis (IAC) among patients admitted to ICU. METHODS: We performed a case-control study in 26 European ICUs during the period January 2015-December 2016. Patients at least 18 years old who developed an episode of microbiologically documented IAC during their stay in the ICU (at least 48 h after admission) served as the case cohort. The control group consisted of adult patients who did not develop episodes of IAC during ICU admission. Matching was performed at a ratio of 1:1 according to time at risk (i.e. controls had to have at least the same length of ICU stay as their matched cases prior to IAC onset), ICU ward and period of study. RESULTS: During the study period, 101 case patients with a diagnosis of IAC were included in the study. On univariate analysis, severe hepatic failure, prior receipt of antibiotics, prior receipt of parenteral nutrition, abdominal drain, prior bacterial infection, anastomotic leakage, recurrent gastrointestinal perforation, prior receipt of antifungal drugs and higher median number of abdominal surgical interventions were associated with IAC development. On multivariate analysis, recurrent gastrointestinal perforation (OR 13.90; 95% CI 2.65-72.82, p = 0.002), anastomotic leakage (OR 6.61; 95% CI 1.98-21.99, p = 0.002), abdominal drain (OR 6.58; 95% CI 1.73-25.06, p = 0.006), prior receipt of antifungal drugs (OR 4.26; 95% CI 1.04-17.46, p = 0.04) or antibiotics (OR 3.78; 95% CI 1.32-10.52, p = 0.01) were independently associated with IAC. CONCLUSIONS: Gastrointestinal perforation, anastomotic leakage, abdominal drain and prior receipt of antifungals or antibiotics may help to identify critically ill patients with higher probability of developing IAC. Prospective studies are needed to identify which patients will benefit from early antifungal treatment.

Thirty Years of Changes and the Current State of Swedish Animal Welfare Legislation.

Sweden is often seen as a leading country in animal welfare and legislation, but some recent amendments to the legislation are perceived as relaxing animal welfare requirements in order to improve the competitiveness of the relevant industry and of farmers. In this study, we analysed the suggested and actual changes in the Swedish national animal welfare regulations relating to horses, cattle and pigs between 1988 and 2019 and the consequences for the intended animal welfare level. The regulations and amendments, including the proposals, the written motivations, the stakeholders' written responses to the proposed amendments and the final amendments, were scrutinised in detail. A sample of 77 requirements was then selected to assess whether and how the animal welfare level was affected by these legislative changes. The results indicated that the animal welfare protection level for cattle, pigs and horses increased overall during the 30-year period, but that a number of specific requirements had been relaxed to meet objectives other than animal welfare. It was more difficult to determine whether animal welfare improved in practice during the same period, due to the lack of systematic evaluations of the consequences of amending the regulations. Future evaluations are needed to evaluate the outcome of new legislative requirements and to monitor whether they serve their purpose in practice.

Managing Animal Welfare in Food Governance in Norway and Sweden: Challenges in Implementation and Coordination.

A key issue in food governance and public administration is achieving coordinated implementation of policies. This study addressed this issue by systematically comparing the governance of animal welfare in Norway and Sweden, using published papers, reports, and legal and other public information, combined with survey and interview data generated in a larger research project (ANIWEL). Governing animal welfare includes a number of issues that are relevant across different sectors and policy areas, such as ethical aspects, choice of legal tools, compliance mechanisms and achieving uniform control. Based on the challenges identified in coordinating animal welfare in Norway and Sweden, relevant organisational preconditions for achieving uniform and consistent compliance were assessed. The results showed that Sweden's organisation may need more horizontal coordination, since its animal welfare management is divided between multiple organisational units (Swedish Board of Agriculture, National Food Agency and 21 regional County Administration Boards). Coordination in Norway is managed solely by the governmental agency Norwegian Food Safety Authority (NFSA), which has the full responsibility for inspection and control of food safety, animal health, plant health, as well as animal welfare. Thus, Norway has better preconditions than Sweden for achieving uniformity in animal welfare administration. However, in Norway, the safeguards for the rule of law might be an issue, due to NFSA acting as de facto "inspector", "prosecutor" and "judge".

The 3Rs in animal welfare bodies at Swedish universities - knowledge, attitudes, implementation.

The implementation of the 3Rs (replacement, reduction and refinement) is emphasized in EU Directive 2010/63. The task of the animal welfare bodies (AWB) is to strengthen animal welfare and develop the 3Rs at research animal facilities. In 2016, we surveyed the knowledge on, attitudes towards and implementation of the 3Rs within AWBs at eight major Swedish universities. Based on responses of 34 closed-ended questions from 44 of 90 AWB members, the overall attitude towards the 3Rs was positive. AWB members did not believe that the 3Rs slow down innovation or result in increased costs, and refinement was considered beneficial for research quality. AWB members were particularly positive towards refinement questions in the survey. A majority of the AWB members predicted that alternative methods will never replace animal use. Researchers as a group represented in the AWBs were significantly less positive towards the 3Rs compared to the group of veterinarians. The tasks of the AWBs, e.g., giving advice on the 3Rs and following up on animal use in projects, were often not carried out in the AWB or not known by the respondents. Our results indicate a need for more practical and regulatory guidance and support to the AWBs. To reach the goal of the EU Directive to phase out animal use in research and education, we suggest that technical expertise in replacement techniques is included in the AWBs. We emphasize the need to strengthen the awareness of the 3Rs among researchers at Swedish universities.

Assessing Animal Welfare and Farm Profitability in Cow-Calf Operations with Stochastic Partial Budgeting.

The societal demand for good farm animal welfare (FAW) has increased over time. Yet, very little is known about the economic consequences of improvements in FAW in cow-calf operations. This study investigates on-farm economic consequences of improved FAW measures in cow-calf operations. It uses a stochastic partial budgeting approach to examine the relationship between contribution margins and improvements in FAW in terms of increased space allowance for a typical Swedish cow-calf operation, as compared to current practices. In the current practice, a cow should be given at least 5 m2 and the calf 2.2 m2. We found that a 0.5 m2 increase in space allowance per calf (achieved by a corresponding reduction of herd size) was associated with a 6.9 to 18.7% reduction in contribution margins in the short term. Our analysis does not include possible indirect gains like decrease in disease incidence and enhanced non-use or 'soft' values associated with increased FAW. However, our analysis indicates that high FAW standards can be costly and careful cost-benefit analysis should be a part of decision-making processes regarding FAW standards. Our results also suggest a need for government support payments and/or the development of market mechanisms to stimulate farmers to continue producing livestock-based foods with high FAW.

Responses of Pigs to Stunning with Nitrogen Filled High-Expansion Foam.

Nitrogen gas (N2) delivered in high expansion foam in a closed container could be a feasible method for humanely stunning pigs. This study aimed to evaluate potential aversion in pigs to the N2 foam method and its effect on stun quality. Furthermore, the study aimed to assess potential aversion to the foam itself. Sixty pigs (27.8 ± 4.4 kg) were divided into three treatments and were exposed to either N2-filled foam, air-filled foam, or no foam air. The N2 foam was effective at purging the air from the container and quickly created stable anoxic conditions. The pigs did not show any strong aversive behaviours when exposed to foam. However, they seemed to avoid putting their heads and snouts into the foam when foam levels became high. Escape attempts through the lid also increased when the foam started covering their heads. The mean time to loss of posture was 57.9 s. Based on the results, stunning with the N2 foam technique could be a viable alternative to high concentration CO2 stunning and potentially lead to improved animal welfare at slaughter.

Instagranimal: Animal Welfare and Animal Ethics Challenges of Animal-Based Tourism.

By animal-based tourism, a host of activities offering passive viewing or active interaction with wild, semi-wild or captive animals is included. The multibillion dollar industry is on the rise globally today, offering modes of engagement with animals that trade on increasingly embodied close encounters with non-human animals. As new modes of animal-based tourism proliferate, such as sloth selfies, visiting cat cafes, swimming with sharks and agri-tourism petting zoos, animal welfare standards risk deteriorating. In the following paper, we collate concerns over animal welfare into a discussion on the challenges facing animal-based tourism. Our synthesis is the first to consider the full spectrum of such animal-based tourism: across agri-, hunting, zoo and safari tourism, to name a few, and crossing consumptive and non-consumptive boundaries. A literature review is first provided. Findings are then presented thematically following workshops at an international interdisciplinary symposium of leading tourism, animal welfare, ethics and leisure sciences scholars together with practitioners of the industry. It discusses macrolevel drivers to animal-based tourism as an industry, the problem of cultural relativism and the role of technology in enhancing or promoting the experience. We indicate ways forward toward implementing a compassionate animal-based tourism.

Repeated intrastriatal application of botulinum neurotoxin-A did not influence choline acetyltransferase-immunoreactive interneurons in hemiparkinsonian rat brain - A histological, stereological and correlational analysis.

In Parkinson's disease, dopamine depletion leads to hyperactivity of cholinergic interneurons in the caudate-putamen (CPu). Botulinum neurotoxin-A (BoNT-A) inhibits the release of acetylcholine in the peripheral nervous system and is also thought to act as a local anticholinergic drug when injected intrastriatally. In hemiparkinsonian (hemi-PD) rats, a unilateral intrastriatal injection of 1 ng BoNT-A significantly diminished apomorphine-induced rotation behavior for at least 3 months, the effect fading thereafter. A second intrastriatal BoNT-A application, 6 months after the first one, led to a stronger and longer-lasting, beneficial behavioral reaction. As a single BoNT-A injection was not cytotoxic in the rat striatum and resembled BoNT-A treatment in clinical practice, here, we investigated the structural outcome of repeated intrastriatal BoNT-A injections with respect to striatal volume, the number of choline acetyltransferase-immunoreactive (ChAT-ir) interneurons and of the length of their dendritic arbors, and the numeric density of ChAT-ir BoNT-A-induced varicosities (BiVs). Repeated unilateral intrastriatal BoNT-A application decreased the volume of the injected CPu, but did not significantly change the number of striatal ChAT-ir interneurons. Also, the total dendrite length of ChAT-ir interneurons after repeated BoNT-A application resembled the values in double vehicle-injected hemi-PD rats. In repeatedly BoNT-A-injected hemi-PD rats, the numeric density of ChAT-ir BiVs in the CPu was increased compared with rats only intrastriatally injected once with BoNT-A. Even repeated BoNT-A injections in rat striata did not cause substantial morphological changes in ChAT-ir neuron, except for the increased numeric density of ChAT-ir BiVs.

Animal handling and stress-related behaviour at mobile slaughter of cattle.

By avoiding animal transportation, mobile slaughter may have the potential to reduce animal stress. In a cross-sectional study with elements of cohort design, we investigated relationships between animal handling and stress-related animal behaviours in connection with slaughter at two Swedish slaughter plants: a newly started small-scale on-farm mobile abattoir and a relatively large-scale stationary slaughterhouse. To the stationary plant, the animals were transported on average 99 km from farms, and one third of these animals spent one night in lairage before slaughter. Data were collected during processing of 298 animals at both plants during one year. Stockperson actions and animal behaviours were observed in the driveways (2.4-7.3 m long) to the stun box. Data on season, hour of day, air temperature, animal breed, animal category, animal age, carcass weight and stockperson category (plant or farm) were also collected. We used Spearman rank correlation, principal-component analysis and confirmatory factor analysis to develop a final generalised structural equation model, which contained six variables that represented observed animal backing; turning; slipping; eliminating; vocalising; and violent behaviour, regressed on a latent variable representing animal stress level. Stress level and animal behaviours were also regressed on five variables representing observed stockperson actions (touching, patting or hitting with hand; touching, patting or hitting with a tool; pushing; tail-twisting; and prodding with an electric goad) and on background variables including plant identity. The animal behaviours were modelled as counts per driveway length, clustered on farm identity. Marginal effects of stockperson actions and predictive margins of plants were calculated. The animals displayed backing and violent behaviour (kicking, goring or violent fighting) at significantly lower frequencies at the mobile plant than at the stationary one. In general, stockperson actions were significantly positively associated with animal behaviours, i.e. increased actions were associated with more frequent behaviours. Stockperson moving with tool, pushing and using electric goad were indirectly significantly associated with all animal behaviours via animal stress level. This study shows the importance of adequate cattle handling to limit pre-slaughter stress.

Effects of prophylactic antibiotic-treatment on post-surgical recovery following intraperitoneal bio-logger implantation in rainbow trout.

Bio-logging devices can provide unique insights on the life of freely moving animals. However, implanting these devices often requires invasive surgery that causes stress and physiological side-effects. While certain medications in connection to surgeries have therapeutic capacity, others may have aversive effects. Here, we hypothesized that the commonly prescribed prophylactic treatment with enrofloxacin would increase the physiological recovery rate and reduce the presence of systemic inflammation following the intraperitoneal implantation of a heart rate bio-logger in rainbow trout (Oncorhynchus mykiss). To assess post-surgical recovery, heart rate was recorded for 21 days in trout with or without enrofloxacin treatment. Contrary to our hypothesis, treated trout exhibited a prolonged recovery time and elevated resting heart rates during the first week of post-surgical recovery compared to untreated trout. In addition, an upregulated mRNA expression of TNFα in treated trout indicate a possible inflammatory response 21 days post-surgery. Interestingly, the experience level of the surgeon was observed to have a long-lasting impact on heart rate. In conclusion, our study showed no favorable effects of enrofloxacin treatment. Our findings highlight the importance of adequate post-surgical recovery times and surgical training with regards to improving the welfare of experimental animals and reliability of research outcomes.

Humanely Ending the Life of Animals: Research Priorities to Identify Alternatives to Carbon Dioxide.

: The use of carbon dioxide (CO2) for stunning and killing animals is considered to compromise welfare due to air hunger, anxiety, fear, and pain. Despite decades of research, no alternatives have so far been found that provide a safe and reliable way to induce unconsciousness in groups of animals, and also cause less distress than CO2. Here, we revisit the current and historical literature to identify key research questions that may lead to the identification and implementation of more humane alternatives to induce unconsciousness in mice, rats, poultry, and pigs. In addition to the evaluation of novel methods and agents, we identify the need to standardise the terminology and behavioural assays within the field. We further reason that more accurate measurements of consciousness state are needed and serve as a central component in the assessment of suffering. Therefore, we propose a roadmap toward improving animal welfare during end-of-life procedures.