Lactoferrin modulates oxidative stress and inflammatory cytokines in a murine model of dysbiosis induced by clindamycin.

Antibiotics, specifically clindamycin, cause intestinal dysbiosis, reducing the microbiota with anti-inflammatory properties. Furthermore, clindamycin 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 clindamycin. Six groups of male C57BL/6 mice were treated with saline (control), clindamycin (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 clindamycin 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, clindamycin 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 clindamycin on intestinal cells and their associated immune system.

Identifying transcriptomic profiles in ovine spleen after repetitive vaccination.

Aluminum hydroxide has long been employed as a vaccine adjuvant for its safety profile, although its precise mechanism of action remains elusive. In this study, we investigated the transcriptomic responses in sheep spleen following repetitive vaccination with aluminum adjuvanted vaccines and aluminum hydroxide alone. Notably, this work represents the first exploration of the sheep spleen transcriptome in such conditions. Animals were splitted in 3 treatment groups: vaccine group, adjuvant alone group and control group. A total of 18 high-depth RNA-seq libraries were sequenced, resulting in a rich dataset which also allowed isoform-level analysis. The comparisons between vaccine-treated and control groups (V vs C) as well as between vaccine-treated and adjuvant-alone groups (V vs A) revealed significant alterations in gene expression profiles, including protein coding genes and long non-coding RNAs. Among the differentially expressed genes, many were associated with processes such as endoplasmic reticulum (ER) stress, immune response and cell cycle. The analysis of co-expression modules further indicated a correlation between vaccine treatment and genes related to ER stress and unfolded protein response. Surprisingly, adjuvant-alone treatment had little impact on the spleen transcriptome. Additionally, the role of alternative splicing in the immune response was explored. We identified isoform switches in genes associated with immune regulation and inflammation, potentially influencing protein function. In conclusion, this study provides valuable insights into the transcriptomic changes in sheep spleen following vaccination with aluminum adjuvanted vaccines and aluminum hydroxide alone. These findings shed light on the molecular mechanisms underlying vaccine-induced immune responses and emphasize the significance of antigenic components in aluminum adjuvant mechanism of action. Furthermore, the analysis of alternative splicing revealed an additional layer of complexity in the immune response to vaccination in a livestock species.

Corrugated intimal surface of the ovine aorta: when physiology resembles pathology.

The aortic lumen in healthy animals is characterized by a smooth, whitish surface, but sheep have macroscopic corrugation of the intimal surface in the thoracic aorta (TA). Our aim was to determine if this finding was pathological or physiological. Thirteen sheep aortas were included in this work together with aortas from cattle (n = 3), a goat (n = 1), horses (n = 4), dogs (n = 2), rabbits (n = 2) and a pig (n = 1). A corrugated intimal surface in the TA was seen in all the sheep and the goat but was less evident in the cattle. Histologically, in sheep the TA intimal surface was seen to have multifocal bulging areas that protruded into the lumen. The outer half of the tunica media had numerous, randomly distributed muscle islands that disrupted the arrangement of the elastic lamella, displacing them towards the lumen. We conclude that the intimal corrugation of the TA in sheep is physiological and must not be misinterpreted as pathological.

A case report of acute claudication due to vena cava thromboembolism in a dog naturally infected with Leishmania Infantum.

Canine leishmaniosis is a vector-borne disease caused by Leishmania infantum, and clinical manifestations of infection range from absent or severe to fatal and result from immune-mediated mechanisms. In dogs, the most common clinical signs of leishmaniosis include skin lesions and lymphadenomegaly. However, the presence of other nontypical signs has been described, and diagnosing these cases can be challenging. The aim of the present short communication was to describe the impact of the formation of circulating immunocomplexes due to L. infantum in a dog with leishmaniosis affected by a massive venous thrombus of the caudal vena cava and external iliac veins. On admission, the dog presented bilateral cutaneous vasculopathy of the thigh and renal disease due to L. infantum infection. Two weeks after starting anti-Leishmania treatment based on meglumine antimoniate and allopurinol administration, the animal developed acute claudication of the hind limbs with the presence of a thrombus in the caudal vena cava and the external iliac veins and a high level of circulating immunocomplexes detected by enzyme-linked immunosorbent assay. Exacerbation of the humoral immune response, along with deposition of circulating immune complexes in the tissues and the concurrent presence of kidney and liver damage, might have contributed to an imbalance in haemostasis in this patient. Future studies should evaluate and analyse the pathological mechanisms contributing to thrombosis in dogs with leishmaniosis.

Mannheimia haemolytica-associated fibrinonecrotizing abomasitis in lambs.

Mannheimia haemolytica-associated abomasitis has been clinically described as a cause of sudden death in lambs, but it is poorly characterized. We describe the pathological features of a severe fibrinonecrotizing abomasitis in 3 lambs that died suddenly. All 3 abomasums had a thickened submucosa due to edema and necrotic areas delimited by bands of degenerate neutrophils with slender nuclei (oat cells) and angiocentric distributions. The overlying mucosa was congested. Myriads of gram-negative coccobacilli were observed within the oat cell bands. M. haemolytica was isolated from the abomasum in all 3 animals and was serotyped as A2 in one of them. Pericarditis and pleuritis were observed in 2 of the lambs. Clostridium spp. were isolated in 1 lamb and detected by immunohistochemistry in the 3 animals, suggesting clostridial co-infection. M. haemolytica should be considered among the differential diagnoses of necrotizing abomasitis in lambs.

Feline osteochondromatosis in a 12-year-old feline leukaemia virus-negative cat.

Feline osteochondromatosis is a spontaneous osteocartilaginous exostosis associated with feline leukaemia virus (FeLV) infection or due to a frameshift variant in the exostosin glycosyltransferase 1 (EXT1) gene. Osteochondromatosis was diagnosed in an indoor-only, 12-year-old, neutered female, Russian Blue cat. Radiographs revealed bilateral calcified proliferations in the elbow, costochondral and sternochondral joints, which distorted the normal skeletal structure. Grossly, the proliferated joints presented with consistent, rounded masses, causing complete ankylosis. The main histopathological finding was an osteocartilaginous proliferation composed of multiple irregular islands of well-differentiated hyaline cartilage surrounded and delimited by osteoid tissue. Immunohistochemistry of the osteochondromas, bone marrow and mediastinal lymph nodes, using a primary anti-FeLV gp70 antibody, and FeLV proviral DNA real-time polymerase chain reaction on bone marrow were negative. Sequencing of exon 6 of the EXT1 gene was performed and nucleotide BLAST analysis demonstrated the absence of a frameshift variant. This study reports the only case of spontaneous feline osteochondromatosis in an animal more than 10 years old.

A case of canine intestinal malakoplakia.

Malakoplakia is a rare chronic granulomatous disease usually affecting the urinary bladder and other locations. In humans, the gastrointestinal tract is the second most common location but there are no reports of intestinal malakoplakia in animals. A 10-month-old female French Bulldog was presented with chronic haemorrhagic diarrhoea and anorexia with normochromic-normocytic anaemia and hypoalbuminaemia. Grossly, there was mucosal thickening and ulceration of the caecum, colon and rectum. Microscopically, transmural sheets of foamy macrophages were seen in these tissues. Macrophages were periodic acid-Schiff, vimentin and ionized calcium-binding adaptor molecule 1 positive and contained von Kossa- and Prussian blue-positive Michaelis-Gutmann bodies. Giemsa staining revealed rod-shaped bacterial colonies and fluorescence in-situ hybridization demonstrated Escherichia coli within macrophages. This is the first reported case of intestinal malakoplakia in domestic animals. Pathological features of intestinal malakoplakia share many similarities with ulcerative histiocytic colitis in dogs but it is unclear if they are different forms of the same pathological process or distinct entities.

Identification and characterization of miRNAs in spleens of sheep subjected to repetitive vaccination.

Accumulative evidence has shown that short non-coding RNAs such as miRNAs can regulate the innate and adaptive immune responses. Aluminium hydroxide is a commonly used adjuvant in human and veterinary vaccines. Despite its extended use, its mechanism of action is not fully understood and very few in vivo studies have been done to enhance understanding at the molecular level. In this work, we took advantage of a previous long-term experiment in which lambs were exposed to three different treatments by parallel subcutaneous inoculations with aluminium-containing commercial vaccines, an equivalent dose of aluminium or mock injections. Spleen samples were used for miRNA-seq. A total of 46 and 16 miRNAs were found differentially expressed when animals inoculated with commercial vaccines or the adjuvant alone were compared with control animals, respectively. Some miRNAs previously related to macrophage polarization were found dysregulated exclusively by the commercial vaccine treatment but not in the aluminium inoculated animals. The dysregulated miRNAs in vaccine group let-7b-5p, miR-29a-3p, miR-27a and miR-101-3p are candidates for further research, since they may play key roles in the immune response induced by aluminium adjuvants added to vaccines. Finally, protein-protein interaction network analysis points towards leucocyte transendothelial migration as a specific mechanism in animals receiving adjuvant only.

Superfluorinated Extracellular Vesicles for In Vivo Imaging by 19F-MRI.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.

Exosomes loaded with ultrasmall Pt nanoparticles: a novel low-toxicity alternative to cisplatin.

BACKGROUND: Platinum nanoparticles have been demonstrated to have excellent anticancer properties. However, because of the lack of specificity they must be delivered to the tumor in amounts sufficient to reach the desired therapeutic objectives. Interestingly, exosomes are considered as excellent natural selective delivery nanotools, but until know their targeting properties have not being combined with the anticancer properties of platinum nanoparticles. RESULTS: In this work we combine the targeting capabilities of exosomes and the antitumoral properties of ultrasmall (< 2 nm) platinum nanoparticles as a novel, low toxicity alternative to the use of cisplatin. A mild methodology based on the room temperature CO-assisted in situ reduction of Pt2+ precursor was employed to preserve the integrity of exosomes, while generating ultrasmall therapeutic PtNPs directly inside the vesicles. The resulting PtNPs-loaded exosomes constitute a novel hybrid bioartificial system that was readily internalized by the target cells inducing antiproliferative response, as shown by flow cytometry and microscopy experiments in vitro. In vivo Pt-Exos showed antitumoral properties similar to that of cisplatin but with a strongly reduced or in some cases no toxic effect, highlighting the advantages of this approach and its potential for translation to the clinic. CONCLUSIONS: In this study, a nanoscale vector based on ultrasmall PtNPs and exosomes has been created exhibiting antitumoral properties comparable or higher to those of the FDA approved cisplatin. The preferential uptake of PtNPs mediated by exosomal transfer between certain cell types has been exploited to create a selective antitumoral novel bioartificial system. We have demonstrated their anticancer properties both in vitro and in vivo comparing the results obtained with the administration of equivalent amounts of cisplatin, and showing a spectacular reduction of toxicity.

Light activated pulsatile drug delivery for prolonged peripheral nerve block.

Regional anesthesia is widely used in peripheral nerve block and in neuraxial anesthesia to reduce anesthetics systemic side effects and shorten recovery times. However, when applied as a single injection (e.g., peripheral nerve block) it is limited by the duration of its effect. Herein, we develop a thermoresponsive nanogel based on poly(oligoethylene glycol methacrylate) containing the long-lasting anesthetic bupivacaine, which can be externally activated by using near-infrared light due to the photothermal properties of hollow gold nanoparticles embedded in the nanogel which facilitate its phase transition, triggering drug release at a controlled temperature above body temperature. Bupivacaine in vitro release can be repeatedly triggered to achieve a controlled pulsatile release of the drug due to the reversible nature of the thermosensitive nanogel, achieving a spatio-temporal control of the release. In vivo sciatic nerve block demonstrates that whereas the administered dose of free bupivacaine produces sensory block and impaired motor function for 2 h, the equivalent bupivacaine dose included in the developed release system can significantly prolong its neurobehavioral anesthetic effect for over 6 h. This release system can also be reactivated multiple times by subsequent irradiation cycles without observing detrimental toxicity in the infiltrated tissues.

Transfer of photothermal nanoparticles using stem cell derived small extracellular vesicles for in vivo treatment of primary and multinodular tumours.

The main current challenges in oncology are (1) avoiding systemic side effects in therapy, and (2) developing alternative treatment strategies for metastatic tumours. Nanomedicine was assumed to provide answers to these issues, but delivering enough therapeutic nanoparticles (NPs) to tumours still remains a huge challenge in nanomaterials-based treatments. Extracellular vesicles (EVs) play a key role in cell communication processes and can be combined with nanomaterials to improve their targeting capabilities. In this work, we leverage the ability of EVs derived from stem cells to reach tumour areas successfully, being used as delivery vehicles for nanoparticles acting as hyperthermia agents. Once small extracellular vesicles (sEVs) loaded with NIR-sensitive hollow gold NPs reached primary subcutaneous solid tumours, they were irradiated with a NIR laser and almost complete tumour remission was obtained. More interestingly, those sEV vehicles were also able to reach multinodular areas similar to those on advanced metastatic phases, eradicating most tumour growth regions in multiple cancerous nodules located in the pancreas region.

Analysis of age-related left ventricular collagen remodeling in living donors: Implications in arrhythmogenesis.

Age-related fibrosis in the left ventricle (LV) has been mainly studied in animals by assessing collagen content. Using second-harmonic generation microscopy and image processing, we evaluated amount, aggregation and spatial distribution of LV collagen in young to old pigs, and middle-age and elder living donors. All collagen features increased when comparing adult and old pigs with young ones, but not when comparing adult with old pigs or middle-age with elder individuals. Remarkably, all collagen parameters strongly correlated with lipofuscin, a biological age marker, in humans. By building patient-specific models of human ventricular tissue electrophysiology, we confirmed that amount and organization of fibrosis modulated arrhythmia vulnerability, and that distribution should be accounted for arrhythmia risk assessment. In conclusion, we characterize the age-associated changes in LV collagen and its potential implications for ventricular arrhythmia development. Consistency between pig and human results substantiate the pig as a relevant model of age-related LV collagen dynamics.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Identification of sheep lncRNAs related to the immune response to vaccines and aluminium adjuvants.

BACKGROUND: Long non-coding RNAs (lncRNAs) are involved in several immune processes, including the immune response to vaccination, but most of them remain uncharacterised in livestock species. The mechanism of action of aluminium adjuvants as vaccine components is neither not fully understood. RESULTS: We built a transcriptome from sheep PBMCs RNA-seq data in order to identify unannotated lncRNAs and analysed their expression patterns along protein coding genes. We found 2284 novel lncRNAs and assessed their conservation in terms of sequence and synteny. Differential expression analysis performed between animals inoculated with commercial vaccines or aluminium adjuvant alone and the co-expression analysis revealed lncRNAs related to the immune response to vaccines and adjuvants. A group of co-expressed genes enriched in cytokine signalling and production highlighted the differences between different treatments. A number of differentially expressed lncRNAs were correlated with a divergently located protein-coding gene, such as the OSM cytokine. Other lncRNAs were predicted to act as sponges of miRNAs involved in immune response regulation. CONCLUSIONS: This work enlarges the lncRNA catalogue in sheep and puts an accent on their involvement in the immune response to repetitive vaccination, providing a basis for further characterisation of the non-coding sheep transcriptome within different immune cells.

Detection of aluminium hydroxide-induced granulomas in sheep by computed tomography: A feasible approach for small ruminant lentiviruses diagnosis and research.

Aluminium (Al) hydroxide use as adjuvant induces local formation of long-lasting subcutaneous granulomas in sheep. Macrophages within these granulomas have been identified as a new small ruminant lentivirus (SRLV) replication site in naturally infected animals. Diagnosis of Al hydroxide-induced granulomas in sheep is mostly based on postmortem observations but little information is available on in vivo detection. Computed tomography (CT) is used for studying these reactions in other animal species. To determine if CT could be a tool for in vivo diagnosis and research of subcutaneous Al hydroxide-induced granulomas in sheep. A retrospective survey on thoracic CT scans was performed on 46 adult sheep. Analysis included absence or presence, number and location of subcutaneous nodules. Thoracic CT scans and pathological studies were prescribed to two further sheep. Single or multiple subcutaneous nodules were detected in 26 (56.52%) sheep. One or two nodules per animal were most often observed (36.95%). Size ranged between 1.5 and 4.5 cm. Pre-contrast two-dimensional (2D) CT images showed focal or multifocal increases in subcutaneous tissue thickness. Post-contrast 2D CT images revealed hypointense areas in the centre. Histopathology indicated the presence of granulomas composed by a large number of activated macrophages, surrounding a central core of necrosis. Large intracytoplasmic Al-positive aggregates were demonstrated by lumogallion staining. CT is a useful tool to detect subcutaneous Al hydroxide-induced granulomas in vivo in sheep. CT provides a diagnostic and research tool that can be very useful in future works in Al hydroxide-induced pathology, SRLV infection, or both.

Photosensitization Induced by Heterophyllaea pustulata in Goats: Sequential Development of Skin Lesions.

Five adult Saanen goats received a single oral dose of Heterophyllaea pustulata containing 42.25 µg/kg rubiadin (anthraquinone) and 3 adult goats were untreated controls. All goats were exposed to sunlight and sequential ear skin biopsies were collected before treatment and at 32 hours, 3 days, 8 days, and 15 days after treatment. Changes at 32 hours after dosing included epidermal spongiosis, single cell death and acantholysis, an increased BAX/BCL-2 protein ratio, and dermal edema. Lesions at day 3 included epidermal and adnexal necrosis, crust formation, and acanthosis. Acanthosis, hyperkeratosis, and dermal fibrosis and neovascularization were present at day 15. The pro-apoptotic (BAX)/anti-apoptotic (BCL-2) protein ratio increased at 32 hours, whereas epidermal and dermal PCNA immunolabeling increased between days 8 and 15 after treatment. The cutaneous lesions were consistent with sunlight-induced damage, and the occurrence in treated but not control goats indicates photosensitization.

Worldwide Prevalence of Small Ruminant Lentiviruses in Sheep: A Systematic Review and Meta-Analysis.

Small Ruminant Lentiviruses (SRLV) are highly prevalent retroviruses with significant genetic diversity and antigenic heterogeneity that cause a progressive wasting disease of sheep called Maedi-visna. This work provides a systematic review and meta-analysis of the last 40 years (1981-2020) of scientific publications on SRLV individual and flock prevalence. Fifty-eight publications and 314 studies were included. Most articles used a single diagnostic test to estimate prevalence (77.6%), whereas articles using three or more tests were scarce (6.9%). Serological tests are more frequently used than direct methods and ELISA has progressively replaced AGID over the last decades. SRLV infection in sheep is widespread across the world, with Europe showing the highest individual prevalence (40.9%) and being the geographical area in which most studies have been performed. Africa, Asia, and North America show values between 16.7% to 21.8% at the individual level. South and Central America show the lowest individual SRLV prevalence (1.7%). There was a strong positive correlation between individual and flock prevalence (ρ = 0.728; p ≤ 0.001). Despite the global importance of small ruminants, the coverage of knowledge on SRLV prevalence is patchy and inconsistent. There is a lack of a gold standard method and a defined sampling strategy among countries and continents.

Growth Performance and Clinicopathological Analyses in Lambs Repetitively Inoculated with Aluminum-Hydroxide Containing Vaccines or Aluminum-Hydroxide Only.

Aluminum (Al) hydroxide is an effective adjuvant used in sheep vaccines. However, Al-adjuvants have been implicated as potential contributors to a severe wasting syndrome in sheep-the so-called ovine autoimmune-inflammatory syndrome induced by adjuvants (ASIA syndrome). This work aimed to characterize the effects of the repetitive injection of Al-hydroxide containing products in lambs. Four flocks (Flocks 1-4; n = 21 each) kept under different conditions were studied. Three groups of seven lambs (Vaccine, Adjuvant-only, and Control) were established in each flock. Mild differences in average daily gain and fattening index were observed, indicating a reduced growth performance in Vaccine groups, likely related to short-term episodes of pyrexia and decreased daily intake. Clinical and hematological parameters remained within normal limits. Histology showed no significant differences between groups, although there was a tendency to present a higher frequency of hyperchromatic, shrunken neurons in the lumbar spinal cord in the Adjuvant-only group. Although Al-hydroxide was linked to granulomas at the injection site and behavioral changes in sheep, the results of the present experimental work indicate that injected Al-hydroxide is not enough to fully reproduce the wasting presentation of the ASIA syndrome. Other factors such as sex, breed, age, production system, diet or climate conditions could play a role.

An outbreak of abortions, stillbirths and malformations in a Spanish sheep flock associated with a bovine viral diarrhoea virus 2-contaminated orf vaccine.

Bovine viral diarrhoea virus (BVDV) is a pestivirus that affects both cattle and sheep, causing an array of clinical signs, which include abortions and malformations in the offspring. Manufacturing of modified live virus (MLV) vaccines often includes the use of bovine-derived products, which implies a risk of contamination with viable BVDV. Recently, the circulation of a specific strain of BVDV 2b among Spanish sheep flocks, associated with outbreaks of abortions and malformations, and whose origin was not determined, has been observed. On February 2018, a MLV orf vaccine was applied to a 1,600 highly prolific sheep flock in the Northeast of Spain that included 550 pregnant ewes. In May 2018, during the lambing season, an unusual high rate (72.7%) of abortions, stillbirths, congenital malformations and neurological signs in the offspring was observed. It was estimated that about 1,000 lambs were lost. Three 1- to 3-day-old affected lambs and a sealed vial of the applied vaccine were studied. Lambs showed variable degrees of central nervous system malformations and presence of pestiviral antigen in the brain. Molecular studies demonstrated the presence of exactly the same BVDV 2b in the tissues of the three lambs and in the orf vaccine, thus pointing to a pestivirus contamination in the applied vaccine as the cause of the outbreak. Interestingly, sequencing at the 5'-untranslated region-(UTR) of the contaminating virus showed a complete match with the virus described in the previously reported outbreaks in Spain, thus indicating that the same contaminated vaccine could have also played a role in those cases. This communication provides a clear example of the effects of the application of this contaminated product in a sheep flock. The information presented here can be of interest in putative future cases of suspected circulation of this or other BVDV strains in ruminants.