First Molecular Characterization of Small Ruminant Lentiviruses Detected in Romania.

Small ruminant lentiviruses (SRLVs) are a group of retroviruses that cause multisystem chronic diseases in goats and sheep and lead to production losses in these animals, negatively affecting animal health and welfare. Although molecular characterization of SRLV field isolates has been performed in many countries, there is currently no information on SRLV genotypes circulating in sheep and goats in Romania. Therefore, the main objective of this study was to conduct a molecular and phylogenetic analysis of SRLVs from Romania and determine the degree of genetic relatedness of the obtained sequences to other known SRLV reference strains. A total of 81 sheep lung tissue samples and 41 sheep lung lymph node samples were tested using nested real-time PCR, and samples positive for real-time PCR were used to amplify an 800 bp gag-pol fragment and an overlapping 625 bp fragment of the gag gene. Pairwise DNA distance and phylogenetic analysis showed that the Romanian SRLV strains were closely related to the A2 and A3 strains based on gag-pol sequences and to the A3 and A17 subtypes based on gag sequences. No recombination events were found. Our results revealed that the Romanian sequences have similar epitope patterns to other existing subtypes, although E/K and R/K mutations in epitope 3 were found only in the Romanian sequences, which may have potential value in serological diagnosis. This study is the first report on the genetic characterization of SRLV strains circulating in Romania and provides new information on SRLV heterogeneity. Further detailed studies should be conducted to better understand the divergence of SRLV Romanian strains.

The evaluation of the osteopromoting capabilities of composites based on biopolymers and gold/silver nanoparticles doped bioactive glasses on an experimental rat bone defect.

The most important concept behind using bone scaffolds is the biocompatibility of the material to avoid a local inflammatory response and must have the following properties: osteoinduction, osteoconductivity, angiogenesis, and mechanical support for cell growth. Gold nanoparticles/gold and silver nanoparticles -containing bioactive glasses in biopolymer composites have been used to enhance bone regeneration. These composites were testedin vitroon fibroblast and osteoblast cell lines using MTT tests, immunofluorescence, scanning electron microscopy analysis, andin vivoin an experimental bone defect in Sprague-Dawley rats. Both composites promoted adequate biological effects on human fibroblastic BJ (CRL 2522TM) cell lines and human osteoblastic cells isolated from the human patella in terms of cell proliferation, morphology, migration, and attachment. Most importantly, they did not cause cellular apoptosis and necrosis. According to the histological and immunohistochemical results, both composites were osteoinductive and promoted new bone formation at 60 d. Evidence from this study suggests that the small amount of silver content does not influence negatively thein vitroorin vivoresults. In addition, we obtained accurate results proving that the existence of apatite layer and proteins on the surface of the recovered composite, supports the validity ofin vitrobioactivity research.

Is the Angiostrongylus vasorum infection in domestic dogs underestimated or misdiagnosed? A comprehensive presentation of four lethal cases.

Introduction: Angiostrongylus vasorum (A. vasorum) is a widely distributed gastropod-borne nematode, causing severe cardio-pulmonary disorders in dogs. In Romania, A. vasorum was detected in foxes and serologically confirmed in domestic dogs, but no clinical cases are currently diagnosed. Methods: Four dogs with clinical history of respiratory distress, originating from different geographical regions of Romania, were included in the study. One dog (case 1) was clinically evaluated using thoracic radiology and cardiac ultrasound; examination of feces and PCR were also performed for the etiologic diagnosis. The postmortem exam was performed in the other three cases, followed by parasitological and molecular analyses. Results: In the first case, parasitic pneumonia was suspected based on the radiographic examination of the thorax and the infection with A. vasorum was confirmed by L1 morphological identification and PCR. The main postmortem changes included large, coalescing, dark red areas of pulmonary consolidation (n = 3) and numerous adult nematodes in the pulmonary arteries (n = 2). The histopathological examination of the lungs showed necrotizing and granulomatous pneumonia with severe hemorrhages and chronic pulmonary arterial changes. Intralesional nematodes were seen in all necropsied cases. Additional inflammatory changes related to A. vasorum infection were identified in the brain and tracheobronchial and mediastinal lymph nodes (n = 2). Identification of larvae, histopathology and PCR confirmed the infection with A. vasorum. Conclusions: This study describes the first cases of canine cardiopulmonary angiostrongylosis in domestic dogs in Romania, and focuses on clinical presentation, pathological findings and molecular analysis. Angiostrongylosis should be included on the list of differential diagnoses of canine cardiopulmonary distress and/or haemorrhagic diathesis in Romania and awareness should be raised among clinicians to avoid post-mortem diagnosis in the future.

First report of Crenosoma vulpis in Africa and Eucoleus aerophilus in Algeria.

Introduction: Crenosoma vulpis and Eucoleus aerophilus are widely distributed lungworms infecting carnivores, mainly red foxes, and are localized in the trachea, bronchi and bronchioles. There are no reports from Africa regarding the presence of C. vulpis. The aim of the present study was to report a co-infection with C. vulpis and E. aerophilus in a red fox from Algeria. Materials and methods: In January 2022, a road-killed male juvenile red fox (Vulpes vulpes) was collected from Bouhadjar-Tarf locality and was submitted for a complete parasitological necropsy. Detected nematodes were collected and preserved in ethanol for morphological and molecular identification. Tissue samples were also collected and analyzed by histopathological methods. Results: Collected nematodes were identified as a male C. vulpis and several Eucoleus aerophilus. The histological techniques of the lung tissue did not reveal the presence of any larvae, or lung inflammation. Conclusion: To the best of our knowledge, this is the first report of C. vulpis infecting a carnivore in this continent, highlighting the importance of further studies to update the geographical distribution of C. vulpis. E. aerophilus was first reported in Algeria. Red foxes are important spreaders of parasitic diseases. Further studies are required for a better understanding of its epidemiology across North Africa and other areas overlapping the range of the red fox.

Hemorrhage and Sudden Death in a Cat with Pancreatic Hemangiosarcoma.

A 15-year-old female, indoor, spayed, domestic shorthair cat was presented for post-mortem evaluation with a history of sudden death. A red-brown 3 cm x 4 cm neoplastic mass was identified within the body of the pancreas, along with multiple similar nodular structures within the omentum and liver. Associated lesions included hemoperitoneum, yellow discoloration of the peritoneal wall, and severe anemia. Spindle-shaped neoplastic cells exhibiting malignancy features, which occasionally contained within the cytoplasm haematoidin crystals and/or red blood cells, were observed during cytological examination. Histologically, the neoplastic cells were organized in short streams forming vascular spaces filled with erythrocytes. Immunohistochemically, the neoplastic cells were immunolabeled for vimentin and CD31, supporting the diagnosis of hemangiosarcoma. This report offers a complete post-mortem and histological evaluation of a rare tumor in cats with an unusual location and a comparative assessment of 3 anti-CD31 antibodies.

Curcumin and Vitamin C Attenuate Gentamicin-Induced Nephrotoxicity by Modulating Distinctive Reactive Species.

Gentamicin remains widely used in all age groups despite its well-documented nephrotoxicity; however, no adjuvant therapies have been established to counteract this side effect. Our study aimed to experimentally determine whether curcumin and vitamin C have nephroprotective effects and whether certain reactive species could be used as markers of early gentamicin nephrotoxicity. Wistar adult male rats were evenly distributed into four groups: control, gentamicin, curcumin and gentamicin, vitamin C and gentamicin (gentamicin: 60 mg/kg/day, intraperitoneally, 7 days). We determined renal function (urea, creatinine), oxidative stress (malondialdehyde, nitric oxide, 3-nitrotyrosine, total oxidative stress), and antioxidant and anti-inflammatory status (thiols, total antioxidant capacity, interleukin-10). Nephrotoxicity was successfully induced, as shown by the elevated creatinine levels in the gentamicin group. In contrast, supplementation with curcumin and vitamin C prevented an increase in urea levels while decreasing total oxidative stress levels compared to the gentamicin group. Moreover, vitamin C and curcumin distinctively modulate the levels of nitric oxide and malondialdehyde. Histological analysis showed more discrete lesions in rats that received vitamin C compared to the curcumin group.

A novel therapeutic phosphate-based glass improves full-thickness wound healing in a rat model.

Wound healing is a highly dynamic process and innovative therapeutic approaches are currently developed to address challenges of providing optimal wound care. In this study, phosphate-based glasses in the (CuO)x ·(KPO3 )79.5-x ·(ZnO)20 ·(Ag2 O)0.5 system (CuKPO3 ZnAg), with different CuO/ KPO3 ratios were prepared by melt-quenching technique. Constant Cu concentrations were released from the samples during immersion in Simulated Body Fluid (SBF), while Zn concentrations were slightly decreased over time. Glass surface phosphatation leading to formation of Zn crystalline salts was revealed through spectroscopic techniques. This finding was supported by SEM images that illustrated new compound formation. Subsequent cytotoxicity evaluation on HaCaT Keratinocytes using the indirect MTT cell viability assay revealed a CuO concentration-dependent cytotoxicity profile and excellent biocompatibility at low CuO concentrations, in all CuKPO3 ZnAg glasses. Furthermore, the (CuO)5 ·(KPO3 )74.5 ·(ZnO)20 ·(Ag2 O)0.5 sample (5CuKPO3 ZnAg), demonstrated superior antibacterial potency against S. aureus (ATCC 25923) strain compared to amoxicillin and ciprofloxacin. In vivo full-thickness wound healing evaluation showed a significantly higher regenerative effect of the 5CuKPO3 ZnAg sample, in terms of angiogenesis, collagen synthesis and re-epithelialization compared to non-treated wounds. These findings advance our understanding of the therapeutic perspectives of phosphate-based glasses, showing promising potential for wound-healing applications.

Protective Effects of Taraxacum officinale L. (Dandelion) Root Extract in Experimental Acute on Chronic Liver Failure.

BACKGROUND: Taraxacum officinale (TO) or dandelion has been frequently used to prevent or treat different liver diseases because of its rich composition in phytochemicals with demonstrated effect against hepatic injuries. This study aimed to investigate the possible preventing effect of ethanolic TO root extract (TOERE) on a rat experimental acute on chronic liver failure (ACLF) model. METHODS: Chronic liver failure (CLF) was induced by human serum albumin, and ACLF was induced in CLF by D-galactosamine and lipopolysaccharide (D-Gal-LPS). Five groups (n = 5) of male Wistar rats (200-250 g) were used: ACLF, ACLF-silymarin (200 mg/kg b.w./day), three ACLF-TO administered in three doses (200 mg, 100 mg, 50 mg/kg b.w./day). RESULTS: The in vivo results showed that treatment with TOERE administered in three chosen doses before ACLF induction reduced serum liver injury markers (AST, ALT, ALP, GGT, total bilirubin), renal tests (creatinine, urea), and oxidative stress tests (TOS, OSI, MDA, NO, 3NT). Histopathologically, TOERE diminished the level of liver tissue injury and 3NT immunoexpression. CONCLUSIONS: This paper indicated oxidative stress reduction as possible mechanisms for the hepatoprotective effect of TOERE in ACLF and provided evidence for the preventive treatment.

Bioactive glass-biopolymers­gold nanoparticle based composites for tissue engineering applications.

Biomaterials based on bioactive glass with gold nanoparticle composites have many applications in tissue engineering due to their tissue regeneration and angiogenesis capacities. The objectives of the study were to develop new composites using bioactive glass with gold nanospheres (BGAuSP) and gold nanocages (BGAuIND), individually introduced in alginate-pullulan (Alg-Pll) polymer, to evaluate their biocompatibility potential, and to compare the obtained results with those achieved when ß-tricalcium phosphate-hydroxyapatite (ßTCP/HA) replaced the BG. The novel composites underwent structural and morphological characterization followed by in vitro viability testing on fibroblast and osteoblast cell lines. Additionally, the biomaterials were subcutaneously implanted in Sprague Dawley rats, for in vivo biocompatibility assessment during 3 separate time frames (14, 30 and 60 days). The biological effects were evaluated by histopathology and immunohistochemistry. The physical characterization revealed the cross-linking between polymers and glasses/ceramics and demonstrated a suitable thermal stability for sterilization processes. The in vitro assays demonstrated adequate form, pore size of composites ranging from few micrometers up to 100 µm, while the self-assembled apatite layer formed after simulated body fluid immersion confirmed the composites' bioactivity. Viability assays have highlighted optimal cellular proliferation and in vitro biocompatibility for all tested composites. Furthermore, based on the in vivo subcutaneous analyses the polymer composites with BGAuNP have shown excellent biocompatibility at 14, 30 and 60 days, exhibiting marked angiogenesis while, tissue proliferation was confirmed by high number of Vimentin positive cells, in comparison with the polymer composite that contains ßTCP/HA, which induced an inflammatory response represented by a foreign body reaction. The obtained results suggest promising, innovative, and biocompatible composites with bioactive properties for future soft tissue and bone engineering endeavours.

Effect of Lactobacillus plantarum ACTT 8014 on 5-fluorouracil induced intestinal mucositis in Wistar rats.

Some previous studies reported that probiotics might decrease the severity of chemotherapy-induced mucositis. This study assessed the potential protective effect of Lactobacillus plantarum ATCC 8014 on 5-fluorouracil (5-FU) induced intestinal mucositis in the Wistar rats. The Crl:WI rats were divided into two groups of six animals (F, L) and one group of 5 animals (M). Group L received for 9 days 3.32x109 CFU/ml of Lactobacillus plantarum orally. In the 7th day of the experiment 400 mg of 5-FU was administered intraperitoneally in groups L and F. Group M received only the vehicles. All animals were sacrificed in the 9th day. Eleven histological characteristics of mucositis were quantified from 0 (normal) to 3 (severe) for duodenum, jejunum and colon. Semiquantitative grades measured Toll-like receptor 4 (TLR4) immunopositive cells. The independent groups were analyzed using the Kruskal-Wallis test, Mann-Whitney U test, with a Bonferroni correction for alpha (P≤0.016). In the group F, treated with 5-FU, the most affected areas were the jejunum and the duodenum. The medium score of histological lesions was 27 for jejunum (minimum 25, maximum 32) and 21 for duodenum (minimum 18, maximum 29). Graded microscopic mucosal changes of the jejunum were significantly lower in group L compared with group F (U=0, P=0.009, Mann-Whitney test). The histological changes depicted on the duodenal and colonic mucosa were less severe in group L than in group F, but without reaching the statistical significance (duodenum: U=6, P=0.172, Mann-Whitney test; colon: U=12, P=0.916, Mann-Whitney test). Although the TLR4 immunoexpression was more intense in group L, no significant statistical difference was revealed at duodenum, jejunum or colonic mucosa. Significantly fewer microscopic changes were depicted in L group on the jejunum, suggesting a potential beneficial effect of Lactobacillus plantarum at this level in 5-FU induced mucositis.

Exogenous Jaagsiekte Sheep Retrovirus type 2 (exJSRV2) related to ovine pulmonary adenocarcinoma (OPA) in Romania: prevalence, anatomical forms, pathological description, immunophenotyping and virus identification.

BACKGROUND: Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by exogenous Jaagsiekte Sheep Retrovirus (exJSRV). The prevalence of JSRV-related OPA in Eastern European countries, including Romania is unknown. We aimed to investigate: the prevalence and morphological features of OPA (classical and atypical forms) in the Transylvania region (Romania), the immunophenotype of the pulmonary tumors and their relationships with exJSRV infection. A total of 2693 adult ewes slaughtered between 2017 and 2019 in two private slaughterhouses from Transylvania region (Romania) was evaluated. Lung tumors were subsequently assessed by cytology, histology, immunocytochemistry, immunohistochemistry, electron microscopy and DNA testing. RESULTS: Out of 2693 examined sheep, 34 had OPA (1.26% prevalence). The diaphragmatic lobes were the most affected. Grossly, the classical OPA was identified in 88.24% of investigated cases and the atypical OPA in 11.76% that included solitary myxomatous nodules. Histopathology results confirmed the presence of OPA in all suspected cases, which were classified into acinar and papillary types. Myxoid growths (MGs) were diagnosed in 6 classical OPA cases and in 2 cases of atypical form. Lung adenocarcinoma was positive for MCK and TTF-1, and MGs showed immunoreaction for Vimentin, Desmin and SMA; Ki67 expression of classical OPA was higher than atypical OPA and MGs. JSRV-MA was identified by IHC (94.11%) in both epithelial and mesenchymal cells of OPA. Immunocytochemistry and electron microscopy also confirmed the JSRV within the neoplastic cells. ExJSRV was identified by PCR in 97.05% of analyzed samples. Phylogenetic analysis revealed the presence of the exJSRV type 2 (MT809678.1) in Romanian sheep affected by lung cancer and showed a high similarity with the UK strain (AF105220.1). CONCLUSIONS: In this study, we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPA in sheep. Additionally, we described the first report of atypical OPA in Romania, and to the best of our knowledge, in Eastern Europe. Finally, we showed that MGs have a myofibroblastic origin.