Aluminum (Al) in the form of γ-aluminum oxyhydroxide (γ-AlOOH) nanoparticles has been used as a vaccine adjuvant for nearly a century. At injection sites, animals and humans develop granulomatous inflammation, in which macrophages may contain intracellular crystalline-like structures, so-called crystalloid bodies (CBs), morphologically distinct from the injected adjuvant. This raised the hypothesis that CBs may originate from the in vivo crystallization of γ-AlOOH within macrophages. To test this, six sheep received two subcutaneous doses of a γ-AlOOH-adjuvanted viral vaccine, while six controls received the same antigen without an adjuvant. All injection sites (AlOOH: n = 12; controls: n = 12) were examined by hematoxylin and eosin, modified aluminum hematoxylin, and lumogallion fluorescence. Al and CBs occurred exclusively in AlOOH-vaccinated tissues. The adjuvant stock and vaccine formulations were characterized ex situ using a combination of structural and imaging techniques. Four granulomas containing CBs, collected 133 days postinjection, were further examined using in situ imaging and crystallographic approaches. No crystalline structures resembling CBs were identified in the injected products; only γ-AlOOH nanoparticles were present. In post-mortem granulomas, macrophages contained Al-positive vacuoles filled with nanoparticle aggregates compatible with γ-AlOOH, often adjacent to CBs. Crystallographic analyses demonstrated that CBs were highly ordered γ-Al2O3 microcrystals, and advanced SEM-EDS with elemental mapping similarly distinguished γ-AlOOH nanoparticles and CBs as separate phases. These findings support an in vivo formation of γ-Al2O3 microcrystals from γ-AlOOH nanoparticles within macrophages under physiological conditions. To our knowledge, these findings provide evidence consistent with Al crystallization within mammalian cells and suggest that macrophage phagolysosomal conditions may represent a previously unrecognized biomimetic pathway for γ-Al2O3 formation. This finding may offer a biological model for the development of green and low-energy synthesis strategies for γ-Al2O3, with potential industrial applications as well as relevance to immunotoxicology, pathology, and cell biology in the context of vaccine adjuvants.

Orf virus (ORFV) is a globally distributed zoonotic parapoxvirus that causes a highly contagious mucocutaneous disease in small ruminants. Despite the urgent demand for vaccination-based control, no licensed vaccines are currently available universally. In this study, we generated two recombinant Sendai virus (SeV) vectors expressing ORFV 011 (rSeV-GFP-B2L) and ORFV 059 (rSeV-GFP-059) genes and evaluated their ability to stimulate antiviral responses in vitro. Following the transduction, we assessed transgene expression, innate immune activation, induction of interferon-stimulated genes (A3Z1, OBST2, SAMHD1), and antiviral activity. Both vectors significantly upregulated pattern recognition receptors (TLRs, RIG-I) and type I interferon (IFN-β) genes, with rSeV-GFP-059 inducing the strongest response. Remarkably, OBST2 was robustly upregulated, suggesting a potential role in restricting ORFV replication. Antiviral activity assays revealed a marked reduction in ORFV DNA copies and a mild decrease in ORFV RNA transcription in rSeV-GFP-059-transduced cells, particularly at later time points, accompanied by complete abrogation of the typical cytopathic effect. Collectively, these results demonstrate that SeV-based vectors, particularly rSeV-GFP-059, efficiently prime antiviral immunity and suppress ORFV replication, establishing a promising platform for further in vivo vaccine evaluation in sheep.

AIMS:During the early stages of the COVID-19 pandemic, significant differences in mortality patterns emerged based on sex and geographical regions. While we were studying on the heredity of variants of the Y chromosome, we observed that regional variations in mortality rates appeared to correlate with the geographical distribution of certain variants of the Y chromosome. This observation led us to propose that some genes on the Y chromosome, with an influence on immune responses, may represent a confounding factor in the observed geographical mortality differences. METHODS:In this analysis, we investigate the potential associations between COVID-19 morbidity and disease-specific mortality and specific Y chromosome variants. The study is based on publicly available pandemic data validated by state authorities or presented in scientific literature documented in PubMed and Medline. RESULTS:We find that Y chromosome haplogroups in different populations exhibit wave-like patterns corresponding with persistent global disparities in COVID-19-related mortality. CONCLUSIONS:These findings warrant further research to uncover possible new pathophysiological mechanisms.

The Chilean southern hake, Merluccius australis, is one of the most important marine fishery resources in Chilean Patagonia. The aim of this study was to investigate the compositional changes in M. australis due to alterations in its volatile composition and other parameters over a period of 23 days in order to evaluate its shelf-life storage at chilled temperatures using fish quality parameters. The samples were bought from the main seafood market in Valdivia city, located in southern Chile.First, an organoleptic evaluation of sensory properties was conducted. Then, pH, total volatile basic nitrogen contents (TVB-N), and trimethylamine (TMA) levels, histamine concentration, and total viable counts (TVC) levels as a microbiological parameter were determined. A repeated measures generalized linear model (GLM) and Duncan's test as post hoc test were used to analyze data.The data revealed that pH levels were always over 7 from day 0 (7.31) to 23 (7.72), which could indicate the prevalence of good but not highest quality in specimens at day 0. No significant differences (P = 0.558) in pH values were obtained during all days of storage at lower than 4 °C.Additionally, the study found a progression of volatile bases levels where TVB-N and TMA levels, increased significantly after 7 days of storage in both cases and exceeded the allowable limit to the family Merlucciidae at day 15 and at day 9, respectively. Both could be considered as reliable indicators by freshness in Chilean southern hake under the present scenario, showing the first empirical results at long storage. On the other hand, histamine concentration was below the allowable limit in fish. And total viable counts (TVC) levels as a microbiological parameter, fluctuated and increased gradually during the first 9 days (from 4.18 log cfu/ml to 4.91 log cfu/ml), and increasing significantly after 15 days (5.42 log cfu/ml) and 23 days (6.32 log cfu/ml) of storage, but did not exceed the spoilage regulation thresholds. These findings emphasize the importance of implementing good practices and improving knowledge in the retail market and to consumers to avoid lengthy storage of fresh fish before consumption, to prevent foodborne illnesses.

A new surgical technique using a polyether ether ketone (PEEK) anchoring device for testicle-sparing laparoscopic inguinal hernioplasty in stallions was described in 2023 and is known as the PEEK harpoon technique (PHT). In breeding stallions, it is essential that the surgery is effective in preventing inguinal hernia but also that it does not impair the testicular function. This study aims to evaluate whether the PHT may affect testicular function. To achieve that, changes in the testicular blood flow, sperm production and motility characteristics were assessed 28 days after use of the PHT. Standing laparoscopic hernioplasty using the PHT was performed unilaterally in eight healthy (non-previously herniated) experimental intact males. The contralateral inguinal canals and testicles were used as control. Pre- and post-surgery Doppler ultrasonographic evaluations of testicular perfusion were performed serially. Bilateral castration was performed at 28 days post-surgery, and epididymal sperm were obtained from both testicles to analyze seminal characteristics. No significant differences were identified in regard to testicular perfusion and the sperm characteristics of the control and operated testicles, suggesting that the PHT-based hernioplasty would not compromise testicular function. Further studies evaluating effects over a longer period are needed, but our data indicate that the PHT is suitable for testicle-sparing inguinal laparoscopic hernioplasties in breeding stallions with a history or predisposing factors of inguinal herniation.

.-The aim was to study the presence and importance of solid pollutants found in the digestive system of White Storks Ciconia ciconia in Arag & oacute;n, North-Eastern Spain. Necropsies of a total of 1,550 storks were carried out. A total of 1,045 presented digestive contents. Solid contents found in the oesophagus, proventriculus and ventriculus were identified by their nature and shape and classified into twelve categories, according to material origins. The volume of these materials was measured and classified as Low, Medium and High, relative to the maximum volume of a normal white stork pellet. Solid pollutants were found in 342 birds out of 1,045 examined digestive contents (32.7%). In 142 of these cases (41.5%), two or more categories of solid pollutants were observed in the same animal. Rubber bands were the most frequently found contaminant, detected in 200 (19.1%) ventriculi. Silicone was found in 66 (6.3%) ventriculi. All other materials were much less frequent. Impaction linked to very large quantities of rubber bands (>200ml) was detected in five cases. Digestive obstruction only represented 0.9% of cases but 6.3% of the examined digestive contents showed a High volume of solid pollutants. The occurrence of such solid pollutants in the digestive did not differ with sex or age. The presence of these materials in the digestive system of white storks was associated with foraging at rubbish dumps and possibly was also related to massive intake of the rubber bands that are sometimes abandoned in cropfields. This is a significant environmental problem affecting White Storks that requires better rubbish dump management, improved agricultural practices and greater environmental awareness by the general population.-Gonz & aacute;lez, Ch., de Blas, I. & Luj & aacute;n, L. (2025). Ingested solid pollutants in White Stork Ciconia ciconia in Arag & oacute;n, Spain. Ardeola, 72: 161-175.

Small ruminant lentiviruses (SRLV) are responsible for significant economic losses in sheep and goat farming; however, effective vaccination strategies remain unavailable. This study evaluated the immunogenicity, safety, and protective efficacy of a recombinant Sendai virus vector (SeV) expressing SRLV gag-P25 (rSeV-GFP-P25) in lambs. Twenty-one SRLV-negative lambs were divided into three groups and inoculated intranasally thrice with culture medium (group 1); SeV-GFP (group 2) or rSeV-GFP-P25 (group 3). Lambs were challenged with homologous SRLV at 16 weeks post-first immunization. Clinical and hematological parameters, antibody responses, SRLV viral loads in peripheral blood mononuclear cells (PBMCs) and target tissues, histopathological and histomorphometric analyses, assisted with artificial intelligence, of interstitial pneumonia were assessed. No clinicopathological alterations were observed, except for a transient temperature increase in group 3 post-first immunization. Group 2 showed mild SeV-neutralizing antibodies, while rSeV-GFP-P25 (group 3) induced negligible SRLV-specific antibody responses. Group 3 exhibited higher SRLV DNA copies in PBMCs but lower in most SRLV target tissues compared to control groups, with no SRLV DNA detected in spleen and bone marrow. Histomorphometry revealed reduced alveolar septal thickening in group 3, indicating partial protection against early SRLV-associated interstitial pneumonia. These results warrant further investigation into cellular immunity and long-term protection.

Persistent subcutaneous granulomas form at the injection site following administration of aluminium oxyhydroxide (AlOOH), a widely used vaccine adjuvant. Small ruminant lentiviruses (SRLVs) can infect macrophages within granulomas induced by commercial AlOOH-based vaccines in sheep. The entry of SRLVs into target cells involves the mannose receptor (MR), while catalytic polypeptide-like 3 protein containing Z1 domain (A3Z1) is considered a restriction factor for lentiviral replication. The objective of this study was to investigate the temporal evolution of AlOOH-induced post-vaccination granulomas in sheep experimentally infected with SRLVs. Twenty-four male lambs underwent two identical vaccination protocols and were challenged with SRLVs. Granulomas were detected in vaccinated groups only and progressively decreased in size. At post-mortem examination, 91.3% of the granulomas were recovered. Fistulas were present in granulomas following the second vaccination protocol. Central necrosis was present in 58.0% of granulomas and was associated with the vaccine used. Orthokeratotic keratin was seen within granulomas in 47.1% of the lambs. Considering all granulomas studied, significantly higher expression of MR was found compared with A3Z1. Differences in MR expression were related to the type of vaccine and the time since vaccination. A3Z1 expression was upregulated in granulomas from the infected groups. Macrophage polarization may influence SRLV infection of granulomas. While SRLV infection does not influence the architecture of post-vaccination granulomas, it may modulate their immune microenvironment. Further studies are needed to elucidate the complex interactions between AlOOH-induced granulomas and SRLV infection in sheep.

Background: Simulation-based teaching is increasingly important in veterinary training, especially for learning anatomy and interventional techniques where real-life exposure is limited. Methods: A handcrafted 3D simulator replicating the canine abdominal arterial system was developed and tested with 80 veterinary students and graduates. A quasi-experimental pre–post design evaluated theoretical knowledge, practical skills, and self-perceived competence after a structured training session including anatomical identification, catheter navigation, and the Seldinger technique. Results: Post-training results showed significant improvements in all domains. Theoretical test scores increased by over 25% (p < 0.001), and more than 85% of participants correctly identified target arteries after training. Navigation success rose to 90%, with shorter execution times and fewer errors. Participants reported increased confidence and satisfaction with the realism and educational value of the simulator. Conclusions: The 3D vascular simulator effectively enhanced learning outcomes and confidence in interventional radiology. Its low-cost, handcrafted design makes it a feasible and valuable educational tool for veterinary institutions lacking access to high-fidelity commercial simulators.

Small ruminant lentiviruses (SRLV) cause multisystemic chronic inflammatory disease and significant economic losses in sheep and goats worldwide. However, no vaccines or therapies are currently available. In this study, a recombinant Sendai virus (SeV) vector encoding the SRLV gag-P25 gene (rSeV-GFP-P25) from the EV1 strain was generated using In-FUSION cloning and rescued using the SeV reverse genetic system. Transgene expression and stimulation of innate immunity and interferon-stimulated genes (ovine A3Z1, OBST2 and SAMHD1) were evaluated in ovine skin fibroblasts (OSF) transduced with SeV-GFP and rSeV-GFP-P25. Additionally, to characterize the effect of the SRLV restriction in transduced OSF, the SRLV DNA load was quantified at different times post-transduction and post-infection with strain EV1. Using immunohistochemistry and image analysis, transgene expression and tissue distribution of recombinant P25 were studied in two lambs inoculated intranasally, one with rSeV-GFP-P25 and the other with SeV-GFP. rSeV-GFP-P25 induced efficient and transient transgene expression in vitro and in vivo. Furthermore, OSF transduced with rSeV-GFP-P25 presented upregulation of TLR2, TLR3, TLR6, TLR7, RIG-I, MyD88 and IFN-β, whereas SeV-GFP did not induce TLR6 or IFN-β upregulation. Among the interferon-stimulated genes, OBST2 was significantly upregulated after transduction with rSeV-GFP-P25 compared with the empty vector. SRLV restriction gradually increased and persisted after transduction with SeV-GFP and rSeV-GFP-P25, with OSF transduced three times showing cumulative restriction. Forty-eight hours post-inoculation in vivo, marked P25 expression was observed in ciliated epithelial cells and submucosal macrophages/dendritic cells of the nasal mucosa. This study reinforces the important role of the innate immune response in controlling SRLV infection and suggests that rSeV-GFP-P25 is a potential vaccine candidate against SRLV.

Aluminum oxyhydroxide (AlOOH) is the most widely used vaccine adjuvant, but its known adverse effects prompt the finding of other, safer alternative adjuvants. This study compares in sheep the global performance and safety of vaccine prototypes against bluetongue virus (BTV) formulated with AlOOH, biomimetic apatite nanoparticles (ApNPs), and microcrystalline tyrosine (MCT). Five groups of 6 sheep were included in the study: control, BTV serotype 4 alone, and BTV-4 combined either with AlOOH, ApNPs, or MCT. Adjuvants were fully characterized. Group specific antibodies against BTV-4 were observed in all treatment groups, including BTV-4 alone. After booster inoculation, ApNPs or MCT groups responded immediately, and it was delayed for BTV-4 and AlOOH groups. Comparable neutralizing antibody responses were observed in all treatment groups but were earlier for BTV-4, ApNPs, and MCT groups when compared with the AlOOH group. No significant systemic alterations were observed during the study. The AlOOH group developed more pronounced local reactions that persisted throughout the 133-day study and were evident as post-mortem granulomas. ApNPs and MCT are biocompatible, safer, and viable alternative adjuvants for sheep vaccines. BTV alone might also be suitable for this purpose. This work is the first demonstrating the suitability of biocompatible alternative adjuvants in sheep vaccines.

Nasal carcinomas in dogs are locally invasive neoplasms with a low metastatic rate that pose significant treatment challenges due to their location and aggressiveness. This study evaluates the safety, feasibility, and therapeutic outcomes of computed tomography-guided radiofrequency ablation (CT-guided RFA) in 15 dogs diagnosed with nasal adenocarcinoma. All patients underwent staging and histopathological diagnosis before treatment. CT-guided RFA achieved a significant tumor volume reduction (82.8%) and improvement in clinical signs such as nasal discharge, epistaxis, and respiratory distress, without complications. Post-RFA CT examinations demonstrated a significant decrease in Hounsfield units and tumor volume. This study has shown that CT-guided RFA is an effective cytoreductive option for minimally invasive management of nasal adenocarcinomas in dogs, particularly when traditional therapies like radiation therapy or surgery are not feasible.

Insulinomas are the most common neoplasms of the endocrine pancreas in dogs, leading to persistent hypoglycemia due to inappropriate insulin secretion. The standard treatment is surgical resection, but it carries significant risks, including pancreatitis and diabetes mellitus. This study investigates the efficacy and safety of percutaneous ultrasound-guided radiofrequency ablation (RFA) as an alternative to surgery. A total of 29 dogs diagnosed with insulinoma were treated with RFA, targeting both primary pancreatic tumors and metastases in regional lymph nodes or the liver. Blood glucose levels and tumor size were monitored before and after the procedure. RFA led to a significant increase in blood glucose levels and a reduction in tumor size in all patients, with minimal postoperative complications. The results suggest that RFA is a feasible and effective treatment option for insulinomas in dogs.

Streptococcus suis is a zoonotic pathogen that causes a major health problem in the pig production industry worldwide. Spain is one of the largest pig producers in the world. This work aimed to investigate the genetic and phenotypic features of invasive S. suis isolates recovered in Spain. A panel of 156 clinical isolates recovered from 13 Autonomous Communities, representing the major pig producers, were analysed. MLST and serotyping analysis revealed that most isolates (61.6%) were assigned to ST1 (26.3%), ST123 (18.6%), ST29 (9.6%), and ST3 (7.1%). Interestingly, 34 new STs were identified, indicating the emergence of novel genetic lineages. Serotypes 9 (27.6%) and 1 (21.8%) prevailed, followed by serotypes 7 (12.8%) and 2 (12.2%). Analysis of 13 virulence-associated genes showed significant associations between ST, serotype, virulence patterns, and clinical features, evidencing particular virulence traits associated with genetic clusters. The pangenome was generated, and the core genome was distributed in 7 Bayesian groups where each group included a variable set of over- and under-represented genes of different categories. The study provides comprehensive data and knowledge to improve the design of new vaccines, antimicrobial treatments, and bacterial typing approaches.

Antibiotics, specifically clindamycin (Clin), cause intestinal dysbiosis, reducing the microbiota with anti-inflammatory properties. Furthermore, Clin can induce alterations in the immune responses and oxidative stress. Lactoferrin, among other activities, participates in the maintenance of intestinal homeostasis and reduces dysbiosis induced by antibiotic treatment. The aim of this study was to analyze the effect of native and iron-saturated bovine LF in a murine model of dysbiosis induced by Clin. Six groups of male C57BL/6 mice were treated with saline (control), Clin, native lactoferrin (nLF), iron-saturated lactoferrin (sLF), nLF/Clin, or sLF/Clin. Oxidation caused in the intestinal cells of the ileum of animals subjected to different treatments was analyzed, focusing on lipid peroxidation and protein carbonyl content. The expression of inflammatory mediators was determined by qRT-PCR. Treatment with Clin did not modify lipid peroxidation, but significantly increased protein carbonyl levels up to almost 5-fold respect to the control, an effect that was reversed by orally administering sLF to mice. Furthermore, Clin increased the expression of interleukin-6 and TNF-α by 1- and 2-fold change, respectively. This effect was reversed by treatment with nLF and sLF, decreasing the expression to basal levels. In conclusion, this study indicates that lactoferrin can prevent some of the effects of Clin on intestinal cells and their associated immune system.

Currently, plastic is used being without any limitations. The livestock sector is no stranger to its use. For example, artificial insemination involves the use of different plastic materials, including semen straws and insemination catheters, which increase the levels of waste in our environment. Finding a sustainable solution to avoid plastic materials is a challenge. The aim of this study was to examine different biodegradable materials as alternatives to conventional syringes used for refrigerated equine semen. The materials tested were bamboo, avocado, grass, paper, Kraft paper, wheat, and rice. Bamboo and avocado were selected to continue testing with the refrigerated semen with a lifespan extended by INRA 96®; the rest of the materials did not meet the requirements necessary for preservation. Motility and movement kinetics tests were performed at 24 and 96 h of refrigeration to evaluate semen quality; relative to the control, significantly better results were displayed in regard to the total and progressive motility avocado straws. The kinetic parameters were comparable to the control for both materials and within normal ranges. After performing a microbiological control process for both straws, it was confirmed that there was no bacterial growth in either straw. We can confirm that avocado straws can be used to preserve chilled equine semen, guaranteeing seminal quality while being respectful to the environment.

Stable flies, Stomoxys calcitrans, stand as formidable pests with a global impact, inflicting significant economic losses on the livestock sector. Larval development occurs in diverse substrates, including decomposing plant material and manure, while emerged adults pose a threat through blood-feeding on both animals and humans. Conventional chemical control methods, predominantly reliant on insecticides, not only pose environmental risks but also face challenges of resistance among stable fly populations. To address this pressing issue, we propose an integrated pest management (IPM) strategy for stable fly control. This approach involved a combination of sanitary-cultural practices, animal protection, the release of natural enemies targeting immature stages, and a specialized trapping system for adults. The Stomoxycc® trap, designed for mass trapping of adult Stomoxys, was employed alongside the release of the predatory mite Macrocheles robustulus and two wasp parasitoids, Spalangia cameroni and Muscidifurax raptor (under the commercial brands Biomite® and Biowasp®) on animal bedding as a key component of this IPM strategy. The implementation of this initiative has been undertaken at a significant sanctuary for donkeys and mules in western Spain. In this publication, we present the application and results of the IPM strategy utilized and provide insights into its use as a sustainable and environmentally friendly option for controlling stable fly populations.

The adequate collection of health information at the farm level is the first step in good health management. The objective of this study was to analyze how Spanish fish farms collect their census, disease occurrence, and mortality data through their farm record books and how they use them for reporting to competent authorities. Comparing and contrasting farm books between regions (autonomous communities) revealed a lack of standardization and substantial differences in the reporting. Therefore, this study points out the need to improve and standardize, at the national level, the design of fish farm record books in order to ensure the better collection of farm census data on mortality and disease occurrence, which may facilitate the production of epidemiological reports and the early detection of emerging diseases at the national level.

Summary Background Several studies have shown dental disease to have a high prevalence and severity in donkeys. Limited studies have assessed the possible relationships between poor dental health and the management and health care of donkeys. Objective The aim of this study was to identify relationships between dental health and current management of donkeys in the UK and to provide recommendations to improve practices to help donkey welfare. Study design Observational cross‐sectional survey. Methods A total of 596 donkeys surrendered to The Donkey Sanctuary (TDS) over a period of 30 months were included. Analysis was performed on three categories of information: entrance information submitted by previous owners and welfare professionals, pre‐admission clinical examination records and arrival medical examination data. Results No statistically significant differences were identified between dental health and provision of bedding, or diet types. Agreement between external professionals and experienced TDS staff was weak in reference to recognition of dental pathology, age and body condition score (BCS). Over 25% of donkeys with severe dental pathology also had poor BCS (1–1.5). The majority of donkeys had no indication of previous dental examination. Main limitations Entrance information was not gathered and recorded in a structured manner. Conclusions Regular prophylactic dental care seems to have poor uptake amongst the owners of the study population, but this is perhaps, in part, aligned to the nature of the sanctuary. Owners are yet to take dental health into consideration when allocating a diet or bedding types to their donkeys, presenting a risk of colic/choke in those fed long fibres with an inability to process them adequately. The Donkey Sanctuary should continue to offer accessible means of education to reach both professionals and owners alike, and this may be well served by the implementation of the new, online, Donkey Academy.

Abstract The authors have requested that this preprint be removed from Research Square.