Organic solvents such as dimethyl sulfoxide (DMSO), ethanol, and methanol are widely used as vehicles in in vitro oncology research, yet their inherent toxicities can confound experimental outcomes. This study systematically evaluates the comparative cytotoxicity and transcriptional signatures of cell death pathways induced by these solvents in the canine mammary carcinoma cell line (CMTU27). Cells were treated with concentrations from 0.25% to 5% for 24, 48, and 72 h. Viability was assessed using the XTT assay, and cell death modes were characterized using Hoechst 33342/Annexin V/PI triple-fluorescence staining. Transcriptional alterations of apoptotic (CASP3, BAX, PUMA, ATM, BID), anti-apoptotic (BCL2, MCL1), and DNA damage/cell cycle (P53, P21, NOXA) genes were quantified via RT-qPCR. DMSO exhibited significant time- and dose-dependent cytotoxicity. No 50% viability concentration was reached after 24 h, whereas linear interpolation yielded approximate concentrations of 4.34% and 3.67% corresponding to 50% viability after 48 and 72 h, respectively. Fluorescent imaging revealed staining patterns consistent with apoptosis and secondary necrosis. Transcriptionally, DMSO markedly upregulated CASP3, BAX, PUMA, and ATM and downregulated MCL1, while BID and BCL2 showed concentration-dependent but not consistently significant changes. Conversely, ethanol and methanol maintained viability above 80% up to 5% (IC50 not calculated). PCA modeling revealed distinct spatial dissociation: ethanol predominantly induced genotoxic stress, strongly correlating with the P53, P21, and NOXA vectors, whereas methanol clustered closest to the control, despite showing significant gene-specific transcriptional alterations. In conclusion, our findings suggest that DMSO concentrations should be kept below 1% in CMT-U27 assays to minimize off-target apoptotic effects. Although ethanol showed limited cytotoxicity, it was associated with DNA damage-related transcriptional responses. Methanol exhibited the most favorable cytotoxicity profile among the tested solvents, although significant gene-specific transcriptional responses were observed, suggesting that its suitability as a vehicle solvent for CMT-U27-based in vitro studies should be evaluated according to the experimental endpoint.
Next-generation sequencing has expanded the study of microbial communities, yet the eukaryotic fraction of host-associated microbiomes remains poorly characterized, particularly in non-intestinal matrices. This study aimed to assess eukaryotic community diversity in bile from small ruminants. A total of 400 bile samples from sheep and goats collected at a Portuguese slaughterhouse were analyzed using 18S rRNA PCR and long-read Oxford Nanopore sequencing on 80 pooled samples. Overall, 12.5% (10/80) of pools were found to have eukaryotic elements, and Cryptosporidium-assigned reads were detected in 30% (3/10) of these, all originating from adult sheep. These findings provide molecular evidence of C. parvum in bile, extending its known tissue distribution beyond the gastrointestinal tract, suggesting potential chronic or subclinical biliary carriage and expanding its recognized tissue tropism. This work highlights the utility of long-read 18S rRNA metabarcoding for detecting protozoan parasites in unconventional biological matrices and characterizing eukaryotic diversity in livestock systems.
This study aimed to generate primary three-dimensional (3D) cell cultures derived from spontaneous canine tumors and to compare two culture systems: a ready-to-use bioreactor (VITVO®) and a miniaturized plate-based system (MPBS). A total of 27 tumors, including mainly hemangiosarcomas, oral melanomas, urothelial carcinomas, and osteosarcomas, were collected from three Italian veterinary hospitals. Fresh samples were used to establish 3D cell cultures and to perform histological and immunohistochemical analyses for phenotypic characterization and comparison with primary tumors. Tissue dissociation proved to be a critical step. A manual protocol combining mechanical and enzymatic methods significantly improved cell viability and cellularity compared to automated dissociation, which yielded fewer viable single cells and limited cell survival. Both culture systems successfully supported 3D cell cultures using the same scaffold material. The plate-based system was particularly suitable for small biopsy samples without compromising cellularity, whereas VITVO® was more appropriate for larger tissue specimens. An inverse correlation was observed between the extent of necrosis in primary tumors and the cellularity of the corresponding 3D cell cultures. Immunohistochemical analyses confirmed that most 3D cell cultures retained the phenotypic characteristics of the original tumors, while osteosarcoma models exhibited limited cell maintenance, likely due to stromal predominance. Overall, these findings highlight the importance of optimizing dissociation protocols, scaffold size, and culture conditions to preserve tumor heterogeneity and viability. This workflow provides a platform for generating 3D models with preserved morphological and immunophenotypic concordance with the primary tumors.
BACKGROUND:Pseudomonas aeruginosa is an opportunistic pathogen recognised for its intrinsic and acquired antibiotic resistance, its capacity to establish chronic infections in cystic fibrosis (CF) airways, and its clinical importance in immunocompromised human and animal hosts. While its extensive secretome has been intensively studied, a mechanistically distinct dissemination strategy has emerged: the constitutive production of outer membrane vesicles (OMVs) that concentrate, protect, and deliver virulence cargo to host cells beyond direct bacterial contact. This systematic review evaluates the molecular mechanisms, biological consequences, antimicrobial resistance implications, and therapeutic prospects associated with OMV-mediated virulence in P. aeruginosa infection, and additionally considers the veterinary and One Health relevance of this evidence base. METHODS:A systematic search of PubMed/MEDLINE, Scopus, Web of Science, and EMBASE was conducted from database inception to January 2025 following PRISMA 2020 guidelines. Studies investigating OMV production, cargo characterisation, or biological activity in the context of P. aeruginosa infection were eligible. Risk of bias was assessed using the ToxRTool (in vitro) and SYRCLE tool (in vivo). Inter-rater agreement was quantified by Cohen's kappa (kappa = 0.82). RESULTS:Fifty-eight studies (predominantly in vitro and murine models) met the inclusion criteria. P. aeruginosa OMVs (50-250 nm) carry biologically active type III secretion effectors (ExoS, ExoU), elastase (LasB), alkaline protease (AprA), pyocyanin, quorum-sensing autoinducers (3-oxo-C12-HSL), Hcp1, siderophores, and beta-lactamase enzymes including VIM and OXA-type carbapenemases. OMV-packaged beta-lactamases were shown, principally in co-culture experiments, to confer transferable resistance by degrading beta-lactam antibiotics in the extracellular milieu, shielding susceptible bystander bacteria. OMVs drive biofilm maturation, suppress CF airway innate immunity, and induce TLR4-mediated hyperinflammation. OMV output was consistently increased under antibiotic selection pressure, in the CF mucus environment, and during quorum-sensing activation, although the magnitude of increase varied considerably across experimental conditions and should be interpreted qualitatively rather than as directly comparable pooled estimates. Therapeutic strategies targeting OMV biogenesis, cargo neutralisation, and OMV-based vaccine platforms are evaluated, and remain at a predominantly preclinical stage of development. CONCLUSIONS:P. aeruginosa OMVs constitute a versatile virulence amplification platform that contributes to tissue destruction, immune evasion, biofilm consolidation, and antibiotic resistance dissemination. Disrupting this multi-functional delivery system represents a promising, though still largely preclinical, antibiotic-independent therapeutic direction for managing refractory P. aeruginosa infections, particularly in CF and critical care settings. Extension of this evidence base to animal-associated P. aeruginosa infections and One Health surveillance is identified as an important priority for future research.
Brucellosis remains one of the most prevalent zoonotic diseases worldwide, causing substantial economic losses and significant human morbidity. Despite decades of research, no licensed human vaccine against Brucella infection exists, and current veterinary vaccines exhibit considerable safety limitations including residual virulence, pregnancy complications, and diagnostic interference. Here we report the development of a multi-epitope messenger RNA (mRNA) vaccine candidate targeting Brucella melitensis, the most pathogenic species responsible for human brucellosis. Using an integrated immunoinformatics pipeline, we screened ten outer membrane proteins (OMPs) and identified Omp25, Omp31, and BP26 as the most immunodominant antigens. A fusion construct incorporating 24 HLA class I and 31 HLA class II predicted epitopes achieved 87.3% global HLA population coverage. Codon optimization improved the codon adaptation index from 0.55 to 0.93. Molecular dynamics simulations over 200 ns confirmed structural stability of the vaccine-TLR4 complex, with a binding free energy of -65.7 kcal/mol. In vitro assays demonstrated robust Th1-biased immune activation, with IFN-gamma reaching 125.6 pg/mL. In a BALB/c mouse challenge model, the mRNA vaccine conferred 80.0% protection, comparable to the live-attenuated S19 vaccine (92.3%) but without associated safety risks. These findings establish a promising platform for Brucella vaccine development warranting further evaluation in large animal models and human clinical trials.
Colonic anastomosis is a critical gastrointestinal procedure, with postoperative leakage, adhesions, and stenosis remaining major concerns. OBJECTIVE:To evaluate the healing progression of end-to-end colonic anastomosis using the modified continuous Gambee technique in rabbits through clinical, macroscopic, and radiographic assessment at different intervals. METHODS:Fifteen adult male rabbits were randomly assigned using a simple random drawing method to evaluation intervals of 7, 14, and 28 days (n = 5 each). After resection of a 2 cm colonic segment, anastomosis was performed using a modified continuous Gambee pattern with 5-0 polydioxanone suture. Three rabbits developed fatal anastomotic leakage before scheduled evaluation and were replaced according to the approved protocol. Evaluations included clinical monitoring, weight changes, leakage testing, adhesion scoring, and radiographic stenosis measurement. RESULTS:Fatal leakage occurred in 3/18 rabbits (16.7%), with necropsy confirming anastomotic dehiscence and local contamination. No macroscopic leakage was detected in surviving rabbits. Adhesion scores showed no significant differences among intervals (P > 0.05). Radiographic stenosis significantly decreased from Day 7 (49.20 ± 2.92%) to Day 14 (22.38 ± 3.17%) and Day 28 (15.94 ± 1.26%) (P < 0.001). Surviving animals showed return of normal activity and feeding with postoperative weight reduction. CONCLUSION:The modified continuous Gambee technique was associated with progressive reduction of radiographic stenosis and acceptable macroscopic healing findings among surviving rabbits. However, fatal anastomotic leakage occurred, and the absence of a comparative group limits conclusions regarding the contribution of this technique to the observed healing outcomes.
A 15-year-old intact male Miniature Dachshund presented with severe hematuria. Clinical evaluations of this dog raised the suspicion of a malignant bladder tumor, and corneal stromal opacity was observed in the left eye. Because of the critical condition, the dog died on the day of presentation, after which a necropsy was performed. Necropsy and histopathological examinations confirmed urothelial carcinoma (UC) in the bladder. Moreover, the corneal stromal opacity represented a metastatic lesion of UC, and similar metastatic lesions were identified at 13 other sites. The UC in this dog was considered clinically highly malignant based on its rapid clinical course and the presence of multiple metastatic sites. This highly malignant nature may have contributed to the corneal stromal metastasis. This is an extremely rare case of corneal stromal metastasis from canine UC.
BACKGROUND:Effective disinfection is crucial to good hygiene practices and biosecurity in veterinary settings. Information about disinfectants and their use in veterinary practices in sub-Saharan Africa is scarce. This study aimed at mapping available veterinary disinfectants for developing a prototype digital disinfectant database in six sub-Saharan African countries (Democratic Republic of the Congo - DRC, Ethiopia, Ghana, Senegal, Nigeria, and Uganda) to support biosecurity and hygienic practices. METHODS:Data on disinfectants available for veterinary use in the six countries were collected between 12/2024 and 11/2025 from various sources (pharmacies, hospitals, veterinary service centres, farmers, companies and registration authorities) and used to create a database of disinfectants. An algorithm-based web application was developed, into which disinfectant data was integrated. RESULTS:Ethiopia and Ghana had the lowest diversity of disinfectants (n = 7 and 11, respectively), while Nigeria had the highest diversity (n = 33), followed by Uganda (n = 23), Senegal (n = 19) and DRC (n = 15). Among 108 total disinfectants of these countries, 20% were chlorines, 19% quaternary ammonium compounds (QACs), 17% aldehydes and 17% alcohols. Alcohols predominated in Senegal (58%), chlorines in DRC (27%), QACs in Nigeria (27%) and aldehydes in Uganda (22%) and Ghana (36%), with chlorines and QACs being common to all countries. The created web-based disinfectant database has many functions for selecting a local disinfectant based on animal species, disinfection purpose and item to be disinfected. CONCLUSION:The study findings revealed marked cross-country diversity variation in veterinary disinfectants and gaps. The developed web-based database represents a step towards supporting veterinary hygienic practices in the study countries.
This study investigated the occurrence of pathogenic Leptospira in urban and rural bats from São Paulo State, Brazil. Kidney samples from 203 bats representing 13 species were screened by real-time PCR targeting the lipL32 gene. Positive samples were further characterized by conventional PCR targeting the lipL32 gene followed by DNA sequencing. Spatial analyses were performed to evaluate possible associations between Leptospira detection, cattle density, and land-use characteristics. Leptospira DNA was detected in 42.6% (43/101) of rural vampire bats (Desmodus rotundus), whereas all 102 urban non-hematophagous bats tested negative. DNA sequencing identified Leptospira borgpetersenii and Leptospira interrogans in 14 positive samples. No significant associations were observed between infection rates and the evaluated environmental variables. The high frequency of pathogenic Leptospira detected in vampire bats highlights their potential ecological association in rural ecosystems and supports further investigations into their role in the ecology and transmission dynamics of leptospirosis.
Leishmaniosis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. It affects humans, canids, and rodents in endemic areas and is transmitted through the bites of infected sand flies. This study aimed to estimate the prevalence of equine leishmaniosis in horses from Tiaret Province, northwestern Algeria, an area endemic for canine leishmaniosis, and to identify the main risk factors associated with infection. The study included 75 local Barb horses and employed molecular and serological techniques for the detection of Leishmania infection. Among the sampled animals, 71 tested positive by at least one diagnostic method, corresponding to an overall prevalence of 94.6%. Horses from the southern part of the province were 75% less likely to be infected than those from other areas (odds ratio [OR]: 0.259, 95% confidence interval [CI]: 0.067-1.004, p < 0.05). These findings provide the first evidence of equine leishmaniosis in northwestern Algeria. Furthermore, the exceptionally high prevalence observed in Tiaret Province suggests intense Leishmania transmission in the area, potentially posing a substantial risk of infection to other susceptible hosts, including humans and dogs. Consequently, the implementation of effective surveillance and control measures is essential to reduce the risk of transmission.
INTRODUCTION:Poor saddle fit can cause back pain and lesions in horses, highlighting the need for preventive therapies. OBJECTIVES:To evaluate the effects of chiropractic treatment on the backs of Mangalarga Marchador horses using infrared thermography to monitor thermal changes associated with saddle-induced overload. METHODS:Physiological, environmental, thermographic, and chiropractic parameters were evaluated in 13 horses subjected to a standardized 25-min exercise session. Animals were assigned to either a control group (GC, n = 6) or a chiropractic treatment group (GT, n = 7). Assessments were conducted at five time points: pre-exercise (M0), immediately post-exercise (M1), 1 h post-exercise (M2), 1 h post-chiropractic assessment (M3), and 48 h post-assessment (M4). Saddles were also evaluated thermographically before (S0) and after (S1) exercise. Data were analyzed using repeated-measures ANOVA. RESULTS:No differences were observed between the groups in the clinical variables evaluated. Mean dorsal and dorsal-midline temperatures were higher in GT at M1; in both groups, these values were higher at M0 than at M3, suggesting a thermal reduction over time. Saddle thermography showed an average increase of ∼1 °C in quadrant 5, and 31% of images exhibited asymmetries and tunnel contact with the horses' dorsal midline. A chiropractic evaluation revealed vertebral subluxation complexes in all horses, primarily in the cervical and lumbar regions. CONCLUSION:Infrared thermography identified saddle-related thermal asymmetries, with no changes in dorsal thermographic patterns after a single chiropractic session. Findings should be interpreted considering the study design and sample size.
Heat stress disrupts intestinal barrier integrity, immune homeostasis, and growth in rabbits, a species highly sensitive to thermal challenges. Microbiota-targeted interventions represent a potential strategy to mitigate these effects. This study evaluated the protective effects of a probiotic (Limosilactobacillus reuteri), a prebiotic (inulin), a postbiotic (reuterin), and a synbiotic (reuterin + β-galacto-oligosaccharides) under natural summer conditions. Rabbits (n = 6 per group) were assessed for thermoregulation, growth, intestinal barrier markers (jejunal zonula occludens-1 [ZO-1], plasma D-lactate), oxidative stress (malondialdehyde [MDA], superoxide dismutase [SOD], catalase [CAT], glutathione [GSH]), inflammatory signalling (TLR4-MyD88-NF-κB, TNF-α, IL-6, IL-10), and gut microbiota composition. All treatments improved thermoregulation, growth, barrier integrity, and redox balance, while suppressing pro-inflammatory signalling, with the synbiotic showing the most consistent benefits, including enrichment of beneficial microbes. In silico analyses indicated that inulin and reuterin interact with key immunoregulatory and metabolic proteins, supporting their functional bioactivity. Mechanistically, these interventions restore intestinal homeostasis by reshaping the microbiota, attenuating endotoxin-driven activation of the TLR4-MyD88-NF-κB axis, strengthening tight junctions, and enhancing antioxidant defenses, thereby reducing epithelial permeability and systemic inflammation. Synbiotic and reuterin-based treatments show the strongest effects, likely via synergistic modulation of host-microbe interactions and intracellular signalling pathways that stabilize barrier integrity, immune balance, and metabolic performance under heat stress.
Occipital malformation (OM) is a congenital cranial abnormality predominantly affecting brachycephalic dogs, frequently associated with atlanto-occipital overlap (AOO), ventriculomegaly, and open fontanelle (OF). This study aims to evaluate the associative value of OF as a marker for OM, ventriculomegaly, AOO, and incomplete cranial ossification using multidetector computed tomography (MDCT). A retrospective observational study was conducted on 399 client-owned dogs with various breeds and skull types that underwent head CT imaging between November 2022 and November 2024. Dogs were classified by skull morphology. Associations between OF and structural abnormalities were determined using odds ratios (OR) and diagnostic performance metric analyses. OF demonstrated strong associations (p < 0.01) with AOO (OR = 56.43, Sp = 99.55%, Sn = 20.34%, PPV = 97.30%, NPV = 61.05%), OM (OR = 43.30, Sp = 99.22%, Sn = 25.35%, PPV = 94.74%, NPV = 70.64%), and incomplete parietal ossification (OR = 34.82, Sp = 99.60%, Sn = 11.97%, PPV = 94.44%, NPV = 67.19%). Significant associations were also found with incomplete occipital ossification (OR = 20.99, Sp = 80.76%, Sn = 83.33%, PPV = 26.04%, NPV = 98.35%) and ventriculomegaly (OR = 5.38, Sp = 72.10%, Sn = 65.57%, PPV = 19.84%, NPV = 95.60%). Dogs with OF and concurrent OM or AOO exhibited a high prevalence of ventriculomegaly, most commonly of moderate severity. The highest proportion of moderate ventriculomegaly (72.22%) occurred in dogs with concurrent AOO. In conclusion, OF demonstrates a strong association and high specificity for concurrent cranial abnormalities, such as OM and AOO. MDCT provides an effective, non-invasive modality for early detection, enabling timely clinical management to prevent neurological deterioration.
Romanowsky-stained smears (RSSs) can be used to immunophenotype lymphoid neoplasms by immunocytochemistry (ICC). This technique typically involves incubating the antibodies separately. However, there is no description of a manual double-staining ICC (dsICC) to characterize lymphocytes in canine samples. This study aims to develop, optimize, and validate a dsICC assay for two lymphoid markers on canine RSSs. An initial antibody selection phase using four candidate markers for B-cells (CD20, CD21, PAX-5, CD79a) and two candidate markers for T-cells (CD3ε, CD5) was performed on unstained and RSSs. Further, CD20 and CD3ε were applied to RSSs for protocol development and optimization. Ultimately, the technique was applied to an independent set of 28 cytological preparations of canine lymphoid neoplasms for validation. The dsICC interpretation was compared with the immunophenotype provided by immunohistochemistry and/or flow cytometry, and/or to the genotype provided by PCR for antigen receptor rearrangement. The dsICC correctly immunophenotyped 26/28 samples (92.85%), including 19 B-cell lymphomas (19/20; 95%) and 7 T-cell lymphomas (7/8; 87.5%); the two inconclusive cases (7.14%) were one B-cell and one T-cell neoplasm. In most smears, the neoplastic B and T lymphocytes displayed a moderate to strong immunoreaction against CD20 and CD3ε, respectively. Residual non-neoplastic T and B-cells were also immunolabeled. The dsICC showed almost perfect agreement (κ = 0.84) compared to confirmatory results. This immunodiagnostic assay represents a potential alternative in veterinary oncology for canine lymphoma immunophenotyping; it enables diagnosis and immunophenotyping from a single smear and may be suitable for the development of an automated dsICC.
Exophthalmia is an important but poorly documented complication of tropical theileriosis caused by Theileria annulata, especially in neonatal and young calves. It involves forward displacement of the eyeball, periorbital swelling, conjunctival congestion, excessive tearing, and enlarged regional lymph nodes. Systemic signs like fever, anaemia, depression, and weakness often occur. The condition arises through complex pathogenetic mechanisms linked to the schizont stage of the parasite, where infected leukocytes transform into rapidly dividing, invasive cells. These cells infiltrate retro-orbital tissues, creating inflammatory masses that pressure the orbital cavity, resulting in globe protrusion. Simultaneously, inflammatory cytokines such as TNF-α, IL-1, IL-6, and IFN-γ are released excessively, causing endothelial damage, increased vascular permeability, and oedema. Enlarged lymph nodes block venous and lymphatic drainage, worsening fluid build-up and infraorbital pressure, which all contribute to the development of exophthalmia in tropical theileriosis.
