Canine leishmaniasis is a major veterinary issue and also a public health challenge due to its zoonotic potential. In this context, serological evaluation is essential for Canine leishmaniasis management. Several serological alternatives, such as rapid diagnostic tests, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT), are well established. In fact, the capacity of distinct tests and antigens, evaluated by their sensitivity and specificity, to detect disease is normally considered sufficient for diagnosing Canine leishmaniasis. In this context, we evaluated the seropositivity using 8 different serological tests (ELISA with Leishmania recombinant proteins (rK39, LicTXNPx); soluble promastigote Leishmania antigens (SPLA); commercial ELISA test) in 82 clinically suspect animals from Northern Portugal. The obtained serological data originated 50% of inconclusive serological information with a mixture of seropositive and seronegative results for individual animals. Cut-off independent risk groups were then generated from the serological data to evaluate the clustering of the samples. This analysis originated risk groups that correlated with the most seropositive samples, suggesting that this method might be used, in a cut-off independent manner, to improve conventional serological evaluation. Ultimately, given that no test prioritization exists, the use of any single serological test increases the potential for misdiagnosis, along with all associated risks for the dog as well as public health. The use of a cut-off independent analysis has the potential to improve the predictive values of these tests, enabling a more accurate evaluation of the dog’s condition.

Most patients with muscle‐invasive bladder cancer (MIBC) are not cured with platinum chemotherapy. Up‐regulation of nuclear factor kappa light‐chain enhancer of activated B cells (NF‐κB) is a major mechanism underlying chemoresistance, suggesting that its pharmacological inhibition may increase platinum efficacy. NF‐κB signaling was investigated in two patient cohorts. The Cancer Genome Atlas (TCGA) was used to correlate NF‐κB signaling and patient survival. The efficacy of cisplatin plus the NF‐κB inhibitor dimethylaminoparthenolide (DMAPT) versus cisplatin or DMAPT alone was tested in vitro. Xenografted and immunocompetent MIBC mouse models were studied in vivo. Platinum‐naive claudin‐low MIBC showed constitutive NF‐κB signaling and this was associated with reduced disease‐specific survival in TCGA patients. Chemotherapy up‐regulated NF‐κB signaling and chemoresistance‐associated genes, including SPHK1, PLAUR, and SERPINE1. In mice, DMAPT significantly improved the efficacy of cisplatin in both models. The combination preserved body weight, renal function, and morphology, reduced muscle fatigue and IL‐6 serum levels, and did not aggravate immuno‐hematological toxicity compared with cisplatin alone. These data provide a rationale for combining NF‐κB inhibition with platinum‐based chemotherapy and conducting a clinical trial in MIBC patients.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led the medical and scientific community to explore the pathogenesis and clinical manifestations of coronaviruses. In felines, a widespread coronavirus known as feline coronavirus (FCoV) can lead to feline infectious peritonitis (FIP), a highly fatal disease characterised by severe systemic inflammation. Diagnosing FCoV remains challenging due to the limited accuracy of the available methods. The present study introduces the FIP Effusion Index, a novel diagnostic method that combines the albumin-to-globulin (ALB/GLOB) ratio with the delta total nucleated cell (∆TNC) count obtained via flow cytometry using the Sysmex XN-1000V^® analyser in effusions. Samples from cats (n = 50) with suspected FIP were analysed for ∆TNC, with findings showing that a ∆TNC ≥ 2.1 is highly indicative of FIP and a ∆TNC ≥ 4.9 can be considered diagnostic. The FIP Effusion Index enhanced diagnostic precision in our group of samples, achieving 96.3% sensitivity and 95.7% specificity for values ≥ 5.06, and reaching perfect specificity (100%) with 96.3% sensitivity for values ≥ 7.54. This combined approach surpasses the accuracy of individual parameters, establishing the FIP Effusion Index as a superior diagnostic tool for FIP, with potential applications in both veterinary and human medicine for related coronavirus diseases.

Leishmania infantum is the causative agent of canine leishmaniosis (CanL), a zoonotic disease of considerable clinical and epidemiological concern. Quantification of parasite load is valuable for clinical management, particularly in low-parasite-load cases. This pilot study evaluated the correlation between cytological and molecular techniques in bone marrow samples from dogs clinically affected with leishmaniosis. Amastigotes were quantified by two independent observers using light microscopy, and the same samples were analysed by real-time polymerase chain reaction (qPCR) employing Leishmania spp. specific commercial primers. Inter-observer agreement was moderate according to Cohen’s kappa (κ = 0.47) and substantial according to the intraclass correlation coefficient (ICC = 0.63), respectively, confirming measurement reproducibility. A very strong inverse correlation was found between parasite counts and qPCR cycle threshold (Ct) values (Spearman’s ρ = −0.89; p < 0.001). Furthermore, a robust logarithmic relationship (amastigotes/μL = 10^{(−0.158 × Ct + 7.61)}; R² = 0.99994) was established allowing direct estimation of in vivo parasite concentration from molecular data. These preliminary findings suggest that qPCR can serve as a reliable, semi-quantitative tool, offering higher sensitivity in subclinical infections. The integration of molecular quantification with cytological methods enhances diagnostic accuracy and supports personalised therapeutic decision-making, advancing clinical management of CanL in a One Health context.

Brucella canis is a neglected zoonotic pathogen associated with canine reproductive disorders and emerging public health concerns. This study presents the first nationwide systematic review and meta-analysis of Brucella spp. in Portugal, integrated with a 13-year retrospective seroepidemiological investigation (2013–2025) of B. canis in dogs across mainland Portugal and Insular Autonomous Regions. Among 132 canine serum samples, a seropositivity of 23.48% was observed using an immunochromatographic assay confirmed by indirect immunofluorescence (IFAT). Significant associations were identified with seasonality (p < 0.001) and breed (p = 0.001), while sex and age were not statistically significant. Municipal-level analysis revealed marked heterogeneity, with Trofa showing the highest seropositivity (58.82%) and a pooled odds ratio of 11.28 (95% CI: 2.90–43.94; p < 0.001). In parallel, meta-analyses of published data estimated a pooled seroprevalence of 2.42% in animals (95% CI: 1.79–3.13) and 10.57% in humans (95% CI: 8.80–12.47), underscoring the broader burden of Brucella spp. exposure in Portugal. These findings suggest underdiagnosis of canine brucellosis and highlight the need for enhanced surveillance in high-risk breeds and regions. The study reinforces the importance of integrated One Health strategies to improve early detection, control, and prevention of B. canis infection in both veterinary and public health contexts.

The antemortem clinical and clinicopathological diagnosis of feline infectious peritonitis (FIP) remains challenging, particularly when effusions deviate from the classically protein-rich, low-to-moderate-cellularity phenotype. The FIP Effusion Index, defined as the ratio of delta total nucleated cells (ΔTNC) to the albumin-to-globulin (A/G) ratio, was developed and initially evaluated in 50 effusions. Subsequent European Advisory Board on Cat Diseases (ABCD) guidelines recommended evaluation in larger cohorts and in cats with confirmed FIP. This study retrospectively evaluated the prespecified threshold (≥7.54) in 176 independent feline effusion samples analysed on the Sysmex XN-1000V and classified by effusion FCoV RT-qPCR, without independent confirmation of FIP status. Relative to this molecular reference, the FIP Effusion Index yielded a sensitivity of 97.8% (95% confidence interval [CI], 92.4–99.7%), a specificity of 88.1% (95% CI, 79.2–94.1%) and an area under the receiver operating characteristic curve (AUC) of 0.962 (95% CI, 0.928–0.995). Discrimination exceeded that of ΔTNC alone (AUC, 0.947; ΔAUC, 0.014; p = 0.012) and A/G alone (AUC, 0.887; ΔAUC, 0.074; p = 0.012). In exudates, sensitivity remained 100% (95% CI, 80.5–100%) and specificity was 82.8% (95% CI, 64.2–94.2%). Among reference-negative effusions, false-positive classification was more frequent in septic than in non-septic samples (6/19 vs. 4/65; odds ratio, 7.04; 95% CI, 1.74–28.53; p < 0.01). These findings demonstrate that the prespecified FIP Effusion Index threshold retained very high sensitivity and strong discrimination for molecular-reference positivity across a broader spectrum of feline effusions. Future studies should evaluate the FIP Effusion Index within a sequential in vivo diagnostic algorithm, using negative results to support rule-out and positive results to prompt confirmation by FCoV antigen detection using immunocytochemistry and/or immunohistochemistry.