Abstract Background Identifying evidence of peri-mortem diarrhoea during necropsy is important for both clinical herd management and research. In live pigs, diarrhoea is typically assessed by inspection of faecal pools on the pen floor scoring of faecal rectal samples, including cotton-swabs, but these methods depend on the availability of faecal material and are therefore often not applicable at necropsy. This study aimed to evaluate intestinal content characteristics and perianal faecal staining as post-mortem indicators of peri-mortem diarrhoea. These indicators were compared with cotton-swab faecal scoring using faecal dry-matter content as a reference. Furthermore, associations with enteric pathogens detected by PCR were investigated. Methods and results A total of 145 pigs from a Danish commercial herd were monitored from birth until 10 weeks of age and euthanised in age-stratified batches for post-mortem examinations. Intestinal content from duodenum, jejunum, ileum, caecum, and colon was evaluated for consistency and colour using categorical scales and compared with perianal faecal staining, faecal scores obtained by the cotton-swab method, faecal dry-matter percentage, and pathogen detections in faecal samples by high-throughput real-time PCR. Colonic content consistency and perianal faecal staining were strongly associated, and in all pigs with watery colonic content a perianal faecal staining was observed. Colonic content consistency corresponded well with faecal dry-matter percentage categories, with mucoid, runny and watery consistencies indicating diarrheic cases (sensitivity: 87%, specificity: 55%). Jejunal content consistency showed little to no association with cotton-swab faecal scores, indicating limited value as an indicator of peri-mortem diarrhoea. Pre-weaning piglets predominantly showed lighter (yellow–green), firmer intestinal contents, whereas post-weaning animals displayed darker and looser contents, particularly in the large intestine. Interobserver reliability was high for consistency scoring (Krippendorff’s α > 0.80 across most segments) but low for colour scoring (α < 0.64). Dark colonic content was associated with PCR detection of Lawsonia intracellularis and Brachyspira pilosicoli , while yellow coloration showed an indirect association with detection of Clostridium perfringens α and β2 toxin genes. Conclusions Post-mortem assessment of colonic content consistency represent a promising, practical and reproducible indicator of peri-mortem diarrhoea, but tending to identify a higher prevalence of diarrheic cases compared to diarrhoea defined by dry-matter percentage. Colonic content consistency also showed a strong association with perianal faecal staining. In contrast, jejunal content consistency showed no diagnostic value as an indicator of peri-mortem diarrhoea. Visual assessment of intestinal content colour demonstrated poor interobserver reproducibility, and any observed associations with enteric pathogens should therefore be interpreted with caution. Overall, these findings support the use of colonic content consistency and perianal faecal staining as necropsy-based indicators for detection of peri-mortem diarrhoea.

Several pathogens are known to affect the respiratory tract of pigs resulting in decreased health and welfare. Porcine parainfluenza virus 1 (PPIV1) and swine orthopneumovirus (SOV) have been identified as novel viruses in pigs. The pathogenicity of PPIV1 has been investigated experimentally by one research group, whereas SOV is yet to be studied. In this experimental trial, two groups of eight pigs were inoculated with a European isolate of PPIV1 or a pool of SOV RNA-positive clinical nasal swab material, and one group of four pigs with culture medium only (negative controls). Nasal swab samples were regularly collected to investigate viral RNA shedding, tissue samples for viral RNA and histopathological examinations, and blood samples to investigate seroconversion. All SOV inoculated pigs tested negative for SOV at 4 days post inoculation (DPI) and therefore, four of these pigs were transferred to the group with the PPIV1-infected pigs to assess direct-contact transmission. At DPI 4, two control and four pigs from each of the PPIV1 and SOV groups were euthanized and necropsied. The remaining pigs were euthanized at 14 DPI. No clinical signs, except for nasal discharge, were observed in any of the pigs. PPIV1 RNA shedding was observed from DPI 2-11 with peaks between DPI 4 and 7, and PPIV1 was transmitted horizontally to all direct-contact pigs. The highest viral RNA load was detected in the upper respiratory tract, i.e., nose, upper and lower trachea compared to the lower respiratory tract, i.e., bronchioles, and alveoli. Generally, a chronic tracheitis at 4 DPI, developing into chronic, erosive tracheitis at 14 DPI was observed in the PPIV1 groups and was supported by in situ detection of PPIV1 by RNAscope. Three pigs also developed mild, bronchointerstitial pneumonia at 14 DPI. All PPIV pigs euthanized at 14 DPI seroconverted. In conclusion, these results showed that PPIV1 is a primary porcine respiratory pathogen that causes breakage of the tracheal epithelial barrier and therefore can predispose to secondary infections. SOV's role as a porcine respiratory pathogen remains unknown, since no successful infection was established and it was not isolated in cells either.

Age assessment of wounds is a challenge in both human and veterinary forensic pathology. This study aimed to evaluate the density, dimensions, and circularity of nuclei in experimental and forensic porcine wounds over time by application of software for nucleus detection and characterization. Moreover, to evaluate whether the nuclei density is affected by the location of the wound on the body. HE-stained tissue sections of granulation tissue from 181 porcine wounds (n = 47 pigs) from a porcine excisional wound model were included, together with 30 porcine skin wounds (n = 22 pigs) received for forensic examination. In addition, tissue sections of intact skin from 13 experimental pigs were included as controls. The experimental wounds had healed by second intention and were divided into seven groups based on wound age (5, 10, 15, 20, 25, 30, and 35 days). Forensic wounds were divided into three age groups based on the age assessment stated in the forensic reports (days, > 1 week, or several weeks). Moreover, the forensic wounds, several weeks old, were divided into three groups based on body location (umbilical outpouching, limbs, and ear/tail). All tissue sections were digitized, and nuclei were quantified and measured (area, diameter, and circularity) by application of the InstanSeg extension in QuPath. The density of nuclei in granulation tissue from the experimental wounds showed a significant decrease over time (P < 0.05, η2P ≥ 0.88). In granulation tissue from forensic wounds, the nuclei density also showed a tendency of a time-dependent pattern, but this was not statistically significant (P > 0.05). Nuclei density was significantly lower in wounds located on umbilical outpouchings compared to wounds located on ears/tails (P < 0.05). Nucleus area, diameter, and circularity did show significant differences between age groups (P < 0.05, η2H ≤ 0.03). However, effect sizes were too low and differences too small to be suitable for age assessment of wounds. Digital quantification of nuclei density is promising as a supportive tool for assessing wound age in veterinary forensic pathology. However, nuclei density can be affected by the body location of the wound and should not be used as a standalone method for age assessment.

Determining the age of wounds in both human and veterinary subjects is a critical aspect of forensic pathology. Gross and histopathological evaluations are used for age estimations, however, these evaluations are subjective as they rely on the opinion and experience of the examining pathologist. Therefore, the aim of the study was to create a multidisciplinary approach to discover accurate and objective indicators of wound age in veterinary forensic cases. In the present study we utilized a porcine experimental wound healing model. Granulation tissue from wounds at different ages (5, 10, 15, 20, 25, 30, and 35 days) (n=188) and control skin samples (n=47) from 47 experimental pigs were evaluated macroscopically, histologically, immunohistochemically, combined with digital pathology, and flow cytometry. Granulation tissue thickness peaked on day 10 post wounding. Full epithelialization was observed at the earliest on day 20 and the percentage of wound surface covered by epithelium increased with age. Leukocytes including hemosiderophages, neutrophils and accumulation of macrophages all displayed time-dependent infiltration into the wound bed. CD34 showed no time-dependent expression. CD105 expression peaked on day 15. CD45 showed a time-dependent expression pattern in the superficial part of the granulation tissue, with highest expression on day 5 and a decrease with increasing wound age. The findings of this multidisciplinary approach indicate the potential to achieve accurate and objective wound age estimations in veterinary and human forensic investigations.

EU regulations concerning the evaluation of animals' fitness for transportation are in several ways rather vague, which leaves room for individual interpretation of the legislation with a potential negative impact on animal welfare. In order to guide interpretation of the unclear statements within the legislation, all forensic case files handled during a 10-year period (2014-2023) on Danish pigs and cattle that had been declared unfit for transportation and subsequently reported to the police, were evaluated. A total of 327 case files addressing 428 animals were examined. Most pigs were declared unfit for transportation due to the presence of severe open wounds, whereas lameness was the main reason for cattle being deemed unfit. In conclusion, most wounds rendering both pigs and cattle unfit for transportation measured at least 3 cm in diameter, apart from those located in sensitive areas, e.g., tails. Manifestations rendering pigs unfit for transportation due to lameness were primarily joint lesions, whereas fractures were the dominant cause of lameness in cattle. Therefore, the focus should be on these manifestations when assessing animals for transport fitness.

Avian aspergillosis, caused by Aspergillus fumigatus (Af), lacks sensitive antemortem diagnostics. Existing microbial cell-free DNA (cfDNA) tests are prone to contamination and require a high pathogen load. We hypothesized that infection-induced tissue damage in chickens creates differentially methylated regions (DMRs) in host cfDNA, enabling machine learning (ML) diagnostics. Serum cfDNA samples (n = 124) were obtained from broiler chickens (n = 76) with Af and non-Af infections (Escherichia coli or Gallibacterium anatis) and controls. Oxford Nanopore sequencing enabled DMR detection and ML training. Performance was evaluated using an independent set (n = 49) and 10-repeat Monte Carlo cross-validation (CV) (n = 490 evaluations per test) as quality control. A High Accuracy test (93 DMRs, neural network) achieved 98.0% accuracy (sensitivity 95%, specificity 100%, AUC 0.974, PR-AUC 0.928) in the independent set, with CV accuracy 92.0% [95% CI: 89.7%-94.4%]. A Fast test (35 DMRs, SVM) achieved 81.6% accuracy and CV accuracy 79.6% [74.9%-84.3%]. An In Situ test (5 DMRs, random forest) designed for field deployment achieved 71.4% accuracy and CV accuracy 62.9% [58.7%-67.0%]. Stratified CV accuracy showed 84.6% [65.1%-95.6%] correct classifications for E. coli and 100% [80.5%-100%] for G. anatis. Markers showed high bootstrap stability and predominantly overlapped EMARs and enhancers. In conclusion, we present MethylSense (https://github.com/markusdrag/MethylSense), an automated open-source software. The High Accuracy test achieved 92.0% [89.7%-94.4%] CV accuracy (CV sensitivity 94.5% [91.4%-97.6%], CV specificity 90.3% [87.8%-92.9%]). While validated in chickens, MethylSense is adaptable to other species and pathogens, offering scalable, contamination-resilient diagnostics for veterinary and conservation applications.IMPORTANCEMethylSense is an automated software for training machine learning diagnostics using differentially methylated regions (DMRs) in cell-free DNA from Oxford Nanopore sequencing. We applied MethylSense to develop three Aspergillus fumigatus tests for chickens, each optimized for different clinical scenarios. The High Accuracy test (93 DMRs, neural network) demonstrated 98.0% accuracy, in a blinded test set (n = 49) with sensitivity 95%, specificity 100%, ROC-AUC 0.974, and PR-AUC 0.928. Stratified 10-repeat Monte Carlo cross-validation (n = 490) showed correct classifications of 84.6% [CI: 65.1%-95.6%] Escherichia coli and 100% [80.5%-100%] Gallibacterium anatis infected specificity samples. A Fast test for rapid <1 h sequencing (35 DMRs, support vector machine) achieved 81.6% accuracy (sensitivity 80%, specificity 82.8%). An In Situ test (5 DMRs, random forest) for field deployment via methylation-specific PCR achieved 71.4% accuracy (sensitivity 45%, specificity 89.7%). Bootstrap analysis demonstrated exceptional marker stability (80.6%-100%) with minimal batch effects, confirming robust host-based diagnostics.

Obtaining an accurate age of skin wounds is a diagnostic challenge in forensic pathology. This study aimed to quantify collagen density and proliferation activity in porcine experimental wounds over time using machine learning-based segmentation to provide an objective method for wound age estimation. Tissue sections from porcine experimental wounds (n = 68) were stained with Masson's trichrome stain or immunohistochemically labeled for proliferation activity by Ki67. The experimental wounds were located on the back and 5-35 days old. Collagen and proliferation activity in the wounds were quantified by training and application of neural network pixel and random trees object classifiers. The relative collagen fraction and the collagen ratio between lower and upper wound regions displayed significant time-dependent patterns. The proliferative activity, assessed by the percentage of Ki67-positive cells, was not suitable for age assessment. In conclusion, the application of a neural network pixel classifier trained to differentiate between collagen and cellular components is an objective method for forensic wound age assessment. However, to obtain a higher precision, the method should be used as a supportive tool in combination with other time-dependent markers.

Obesity is a well-recognized risk factor for increased severity following influenza A virus (IAV) infection, likely by promoting meta-inflammation and immune dysregulation, but its effects on antiviral and inflammatory responses remain poorly understood. Using a Göttingen minipig model of diet-induced obesity, we compared antiviral and inflammatory responses before and after IAV infection. Respiratory tract transcriptomics and histopathology revealed no clear obesity-associated immune or inflammatory changes in uninfected minipigs. In contrast, obese pigs showed reduced viral clearance in nasal mucosal tissue accompanied by altered antiviral gene expression pattern 4 days post-infection. Furthermore, cellular infiltration of the respiratory tract was observed in obese pigs after infection. Finally, C-reactive protein concentrations increased significantly in obese pigs, indicating enhanced systemic inflammation in response to respiratory infection. These findings demonstrate that diet-induced obesity in Göttingen minipigs is associated with altered antiviral and inflammatory responses following IAV infection and support the translational value of the obese minipig model.

Birth asphyxia can result in kidney dysfunction, disturbances in systemic electrolytes and fluid balance in newborns. Currently, there is no proven dietary approach to support asphyxiated newborns. This study investigates whether oral plasma supplementation improves kidney function and overall health in asphyxiated newborns. Cesarean-delivered near-term pigs with or without an 8 min intrauterine clamping of the umbilical cord were fed a milk replacer dissolved in water for 24 h in Experiment 1. Pigs were fed 72 h with milk replacers dissolved in either maternal plasma or water in Experiment 2. Blood, urine, and kidney tissue were collected for further analyses. Asphyxia disrupted blood electrolyte balance. And plasma feeding led to higher fluid retention for both asphyxiated and control pigs. Additionally, plasma feeding may also affect kidney development and protect kidneys from asphyxia induced impairments. Birth asphyxia in pigs led to immediate disturbance of electrolyte balance, impaired fluid retention, and kidney impairments. Plasma feeding may improve postnatal newborn hydration and may also improve the condition of kidneys following asphyxia.

BackgroundMicroRNAs, a class of small noncoding RNAs, serve as post-transcriptional regulators of gene expression and are present in a stable and quantifiable form in biological fluids. MicroRNAs may influence intra-articular responses and the course of disease, but very little is known about their temporal changes in osteoarthritis.ObjectivesTo identify miRNAs and characterise the temporal changes in their abundance in SF from horses with experimentally induced osteoarthritis. We hypothesised that the abundance of miRNA would change during disease progression.Study designIn vivo experiments.MethodsRNA extracted from synovial fluid obtained sequentially (Day 0, 28 and 70) from nine horses with experimentally induced osteoarthritis was subjected to small RNA sequencing using the Illumina Hiseq 4000 sequencing platform. Differentially abundant miRNAs underwent further validation and mapping of temporal abundance (Day 0, 14, 17, 21, 28, 35, 42, 49, 56, 63 and 70 days after osteoarthritis induction) by microfluidic reverse transcription quantitative real-time PCR. Bioinformatic analyses were performed to predict potential biological associations and target genes of the differentially abundant microRNAs.ResultsSmall RNA sequencing revealed 61 differentially abundant microRNAs at an early osteoarthritis stage (Day 28), and subsequent reverse transcription quantitative real-time PCR analysis validated 20 of these. Significant biological functions of the differentially abundant microRNAs were apoptosis, necrosis, cell proliferation and cell invasion. Following validation, four microRNAs (miRNA-199b-3p, miRNA-139-5p, miRNA-1839 and miRNA-151-5p) were detected in more than 50% of the synovial fluid samples and had higher abundance in osteoarthritic than in control joints.Main limitationsLimited sample size.ConclusionThis is the first study to determine longitudinal changes in synovial fluid microRNA abundance in an equine model of osteoarthritis. Larger studies are needed in naturally occurring osteoarthritis to interrogate putative changes identified by this study.

Development of new drugs typically involves the identification and validation of molecular inhibitors or promotors of endogenous biological processes. The identification of ligands that can bind the target of interest is typically achieved by screening large libraries of small molecules, using analytical methods that only provide yes/no answers. These methods are only qualitative and often associated with unacceptable amounts of false positives and negatives. Quantitative methods are in general more accurate but time intensive. This is mainly due to repeating measurements of a dilution series in order to generate a titration curve and measure the dissociation constant (Kd). In this work, we introduce Continuous Titration Based Spectral Related Intensity Change (cSPRING), that combines Taylor dispersion analysis (TDA) with ratiometric fluorescence detection to measure a Kd in a single experiment. cSPRING is an in-solution method that reduces the sample preparation time 8-fold and requires only nanograms of protein. We show a good agreement of cSPRING with other quantitative methods for three well-known protein-small molecule interactions with binding affinities ranging from the low nanomolar to high micromolar. In addition, we show that cSPRING is able to measure binding affinities in under a minute, highlighting its efficiency and potential for screening applications.

Introduction:Use of Machine learning (ML) is rapidly expanding in histopathology, offering the potential to reduce interobserver variability and improve quantitative assessment. However, large datasets and computational resources commonly used in toxicology and human medicine are often unavailable to the veterinary pathologist. This study aimed to evaluate the feasibility and limitations of applying supervised ML on histopathological samples with limited training data, exemplified by training an ML model to segment the intestinal wall into its histological layers. Materials and methods:The study included 145 piglets from five age groups (4, 14, 25, 49, and 67 days). Full-wall samples from duodenum, jejunum and ileum were collected post-mortem, stained with H&E and digitized. A three-step ML model was trained on 8-15 images: Step 1 identified tissue, Step 2 segmented mucosa from submucosal layers, and Step 3 separated lamina propria from epithelium. Model performance was assessed by comparing AI-generated areas to manual annotations, calculating relative deviation, categorized agreement levels, Intersection over Union, and Pearson correlation coefficients. Qualitative error analyses were used as directions for future training options. Results:A three-step separation model was successfully developed, but showed a significant amount of age-related performance variation, depicted as larger inaccuracies in samples from the younger age-groups, reflecting additional tissue heterogeneity from immature morphology. Classification errors could be categorized into intrinsic limitations (e.g., thresholding issues in tissue identification) and training deficits (e.g., misclassification of goblet cells and crypt abscesses), of which only the latter category could be corrected by adding additional training data. Conclusion:This study demonstrates the feasibility of ML-based histopathology with limited sample sizes, providing a viable option for veterinary pathologists. Models trained on small datasets require careful supervision, with special emphasis on age-diverse tissue heterogeneity and overfitting. In these cases, ML should be seen as a tool to augment, not replace, expert oversight, ensuring reliable and reproducible quantitative histopathological measures.

Background Globally, the increase in antimicrobial resistance is of great concern. In Denmark, the pig sector is accountable for the majority of antimicrobial usage in animals. As new-born piglets are at risk of developing infectious omphalitis, and many pigs are treated by antimicrobials within the first days of life, an early and accurate diagnosis of the disease is imperative to maintain animal welfare and reduce the antimicrobial usage. The aim of the present study was to compare histopathological and microbiological findings in piglets clinically diagnosed with and without omphalitis during the first three days after birth. Results A total of 98 case piglets with omphalitis and 98 control piglets without omphalitis, based on clinical examinations during the first three days of life, were included. Of the 196 piglets, 79 (38 cases and 41 controls) presented histopathological omphalitis. Post mortem, the volume of the umbilicus was significantly enlarged in cases compared to controls (p = 0.01). Except for a group of bacteria unidentified by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) (p = 0.02), no apparent association was found between histopathologically diagnosed omphalitis and the prevalence of specific pathogens (p > 0.05). The frequency of histopathologically diagnosed omphalitis tended to increase with increasing age in both cases and controls. The frequency of piglets with both histopathological omphalitis and arthritis/synovitis was significantly different among cases and controls (p = 0.05). This was due to all controls, and none of the cases, with arthritis/synovitis presented histopathological omphalitis. Conclusion The clinical differentiation between omphalitis cases and controls did not correspond to the histological diagnosis of omphalitis in zero-to-three days old piglets. An inaccurate clinical diagnosis complicates prudent use of antimicrobials in pig herds. In addition, animal welfare may be hampered in infected piglets due to lack of treatment and in healthy piglets due to the antimicrobial effect on the gut microbiome.

Highly pathogenic avian influenza A virus (HPAIV) H5N1, clade 2.3.4.4b, has emerged as a significant zoonotic threat. H5N1 is widely circulating in wild birds, and an increasing number of spillover events have been observed in a wide range of mammalian species. These cases are primarily reported in countries on the European and American continents. This review describes the likely transmission routes, lesions, and clinical manifestations of HPAIV H5N1 clade 2.3.4.4b in naturally infected mammals, with a focus on the involvement of the central nervous system (CNS). In the analysis, pathological findings were categorized by organ system and host species, which were further divided into terrestrial mammals, marine mammals, and dairy cattle. The most frequently reported clinical manifestations were neurological and respiratory signs in marine mammals and neurological signs and lethargy in terrestrial mammals. Macroscopic and histological lesions were commonly found in the CNS and lungs of terrestrial and marine mammals, while dairy cattle showed mainly gastrointestinal and mammary gland involvement. Immunohistochemistry and reverse transcriptase real-time PCR analyses confirmed high viral loads in brain tissues, indicating a neurological tropism of H5N1 clade 2.3.4.4b. Routes of CNS invasion remain uncertain, though both hematogenous and olfactory nerve pathways are discussed. Recent evidence suggests mammal-to-mammal and vertical transmission, raising concerns for the zoonotic and pandemic potential of this virus. In conclusion, the findings emphasize an urgent need for enhanced surveillance to effectively disclose changes in viral pathogenicity and transmissibility among mammalian hosts.

Background Intranasal vaccines against respiratory viruses are desired due to ease of administration and potential to protect against virus infection of the upper respiratory tract. Methods We tested a cationic liposomal adjuvant delivering the TLR3 agonist Poly (I:C) (CAF (R) 09b) for intranasal administration, by formulating this with SARS-CoV-2 spike trimeric protein and assessing airway mucosal immune responses in mice. The vaccine was further evaluated in SARS-CoV-2 virus challenge models, using mice expressing the human ACE2 receptor and Syrian hamsters. Findings The intranasal vaccine elicited both serum neutralising antibody responses and IgA responses in the upper respiratory tract. Uniquely, it also elicited high-magnitude CD4 and CD8 T cell responses in the lung parenchyma and nasal-associated lymphoid tissue. In contrast, parenteral administration of the same vaccine, or the mRNA1273 (Spikevax (R)) vaccine, led to systemic antibody responses and vaccine-induced CD4 T cells were mainly found in circulation. The intranasal vaccine protected against homologous SARS-CoV-2 (Wuhan-Hu-1) challenge in K18hACE2 mice, preventing weight loss and virus infection in the upper and lower airways. In Syrian hamsters, the vaccine prevented weight loss and significantly reduced virus load after challenge with the homologous strain and Omicron BA.5. Interpretation This study demonstrates that intranasal subunit vaccines containing TLR3-stimulating cationic liposomes effectively induce airway IgA and T cell responses, which could be utilised in future viral pandemics.

Determining the age of wounds is of utmost importance in both veterinary and human forensic pathology. The aim of this study was to design and optimize quantitative polymerase chain reaction (qPCR) primers for use on degraded RNA samples obtained in veterinary forensic cases, hereby ensuring assay robustness. Moreover, the aim was to evaluate if an expression signature, based on optimized short-amplicon primers, was able to differentiate porcine experimental granulation tissue according to age and if this could be used for wound age assessment in veterinary forensic cases. Initially, 12 samples of experimental granulation tissue (n=6) and skin (n=6) from two pigs were deliberately exposed to RNA degrading conditions before being stored in RNAlater. A panel of 24 robust primers were selected based on the intentionally degraded samples. Granulation tissue (5, 10, 15, 20, 25, 30, and 35 days of age) (n=94) and control skin (n=47) from 47 experimental pigs was sampled and stored in RNAlater. Furthermore, granulation tissue and fibrous scar tissue were sampled from 14 veterinary forensic cases. Microfluidic qPCR was performed to evaluate the gene expression of 24 genes. An expression signature of 14 genes reflected the age of the experimental wounds. The 5-day old wounds displayed the biggest divergence from the control skin. As the granulation tissue matured, gene expression gradually approached the levels observed in intact skin. The forensic samples clustered somewhat separately from the experimental samples. In conclusion, granulation tissue from the experimental wounds displayed a time-dependent expression profile based on 14 short-amplicon primers suitable for use with low-quality RNA. However, an expression profile of 14 genes cannot be used as the sole method for forensic age assessments of porcine wounds.

Background: Premature infants are highly susceptible to infections that can lead to sepsis with life-threatening organ dysfunctions. The clinical practice of high parenteral glucose supply in preterm infants can exacerbate infection outcomes through excessive glycolysis-induced inflammatory response. This in turn can affect the health of vital preterm organs, including the brain and kidneys. We hypothesized that reduced parenteral glucose supply to infected preterm newborns may help protect against pathology in these two key organs. Methods: Cesarean-delivered preterm pigs were nourished with high or low parenteral glucose levels (21 % vs. 5 %), infused with Staphylococcus epidermidis or saline, and monitored in heated, oxygenated incubators until 22 h. Blood, brain, and kidney samples were collected for histological, immunohistological, q-PCR, ELISA, and biochemical analyses. Results: Infection led to multiple pathological changes (e.g. edema), increased inflammation and tissue injury (indicated by gene expression data) in both brain and kidneys of preterm piglets. Reduced glucose supply in infected animals alleviated histopathological manifestations in the brain, and reduced neuroinflammation with enhanced M2 microglial phenotype. Reduced glucose supply also decreased plasma creatinine, and the severity of renal edema, tubular vacuolization and dilatation. Multiple genes related to innate and Th1 immunity in both organs were dampened by reduced glucose supply. Correlation analysis showed that renal inflammation was more closely connected to systemic inflammation compared to neuroinflammation. Conclusion: Reduced glucose supply can reduce renal and neuro-inflammation during neonatal infection, thereby protecting brain and kidney health in infected preterm neonates.

Antibiotic treatment of piglets after birth is commonly carried out due to concern for development of omphalitis leading to umbilical outpouchings and/or systemic infections. Among others, the portal of entry for bacterial infections includes the umbilical cord at birth. The aim was to characterise the histological and bacteriological pattern of manifestations in the umbilicus of piglets with omphalitis that died during the suckling period in a Danish herd. A total of 37 piglets found dead or euthanised due to sickness before weaning were included. Histopathological omphalitis was diagnosed in 13 of these piglets, and umbilical lesions and bacteria were most often observed in association with the umbilical blood vessels. Neutrophilic granulocyte infiltrations were observed in association with both umbilical arteries and the vein, occurring most frequently in the arteries. Escherichia coli and Trueperella pyogenes were the most commonly isolated bacteria from piglets with histopathological omphalitis. Omphalitis in piglets was characterized by inflammation and presence of bacteria in the umbilical arteries and to a lesser extent the umbilical vein. Inflammation in urachus was not present.

Several pathogens are known to affect the respiratory tract of pigs resulting in decreased health and welfare. Virological investigations and the use of metagenomic sequencing of samples allowed the identification of new viruses in pigs, such as porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). The pathogenicity of PRV1 has been investigated experimentally by one research group, whereas SOV is yet to be studied. In this experimental trial, groups of weaners were inoculated with a European isolate of PRV1 ( n = 8), a pool of SOV RNA-positive clinical nasal swab material ( n = 8) or culture medium serving as controls ( n = 4). Four days post inoculation (DPI), two controls and four pigs from each of the PRV1 and SOV groups were euthanized and necropsied. All SOV inoculated pigs tested negative for SOV at DPI 4 and therefore four of these pigs were transferred to the stable with the PRV1- infected pigs to assess direct-contact transmission. Nasal swab samples were collected at regular intervals after challenge and blood samples were collected at DPI 0, 4, and 14. At 14 DPI, the remaining pigs ( n = 10) were euthanized and necropsied. Nasal swab samples and tissues from the respiratory tract were examined for PRV1 RNA by reverse transcriptase real- time PCR and blood samples were investigated for PRV1 antibodies by ELISA. Respiratory tissues were also evaluated macro- and microscopically and selected tissues were investigated for in situ detection of PRV1 mRNA by RNAscope. No clinical signs, except for nasal discharge, were observed in any of the pigs. PRV1-shedding was observed from DPI 2 to 11 with peaks between DPI 4 and 7, and PRV1 was transmitted horizontally to all direct-contact pigs. The highest viral load was detected in the upper respiratory tract, i.e. nose, upper and lower trachea compared to the lower respiratory tract, i.e., bronchioles, and alveoli. Generally, a chronic tracheitis at 4 DPI, developing into chronic, erosive tracheitis at 14 DPI was observed in the PRV1 groups and was supported by in situ detection of PRV1 by RNAscope. Three pigs also developed mild, bronchointerstitial pneumonia at 14 DPI. In conclusion, these results showed that PRV1 is a primary porcine respiratory pathogen. Author summary Respiratory diseases in pigs impair porcine health and welfare and are often caused by a complex interaction between pathogens. We investigated the pathogenesis of two recently discovered viruses: porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). An experimental model in weaner pigs showed that PRV1 is capable of efficient replication, horizontal transmission and caused pathological manifestations mainly in the upper respiratory tract. The lesions consisted of chronic, erosive tracheitis in all PRV1 infected pigs, resulting in a breakage of the tracheal epithelial barrier, and a few pigs developed bronchointerstitial pneumonia. These findings confirm that PRV1 should be considered a primary porcine respiratory pathogen, but in contrast, SOV did not result in a successful infection, and the reasons why remain to be elucidated. ![Figure][1] Graphical abstract created with [Biorender.com][2] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes [2]: https://Biorender.com

In Denmark, pigs with wounds on umbilical outpouchings (UO) are deemed unfit for transport, due to welfare concerns, particularly the risk of wound rupture when these pigs are transported. Consequently, these pigs are often killed on-farm thereby affecting sustainability of pork production and farmers’ economy. To gain more knowledge on the impact of transportation on wounds on UOs of pigs, this study examined 96 Danish slaughter pigs with wounds on the UOs and investigated how clinical signs were associated with the distance between the wound surface and the cavity of the UOs measured at necropsy. The pigs originated from three conventional herds and were clinically examined by the herd veterinarian the day before transport to the abattoir. From the abattoir, the UOs were sent for pathological examination at University of Copenhagen where the distance between the wound surface and the peritoneal lining of the cavity of the UOs was measured. The results showed that the maximum measurement of either the length or width of the wound measured clinically was 2.93 cm (σ = 0.97). The mean distance between the wound surface and the cavity of the UOs of 87 slaughter pigs was 2.27 cm, and a total of 94