Serum protein capillary electrophoresis can characterise dysproteinaemia in dogs tested for canine leishmaniosis (CanL) as an integrated clinicopathological phenotype of humoral immune activation. This retrospective laboratory-based study evaluated serum protein electrophoretic patterns in 5544 dogs tested for CanL in Portugal between 2009 and 2026. Anti-Leishmania antibody results were classified into five ordered categories: negative, inconclusive, low positive, positive/high positive and very high positive. Total protein, albumin, alpha-1, alpha-2, beta and gamma fractions, albumin-to-globulin and albumin-to-gamma-globulin ratios were analysed. A separately defined reference subpopulation of 254 clinically healthy seronegative dogs aged ≥ 1 to <4 years was used to establish study-specific non-parametric 95% reference intervals. After excluding inconclusive results, binary analysis included 2454 seronegative and 2910 seropositive dogs. Seropositive dogs had higher median gamma globulins than seronegative dogs (1.5 vs. 0.8 g/dL; p < 0.001), with lower albumin-to-globulin (0.7 vs. 1.0; p < 0.001) and albumin-to-gamma-globulin ratios (1.9 vs. 4.2; p < 0.001). Across the ordinal serological scale, gamma globulins increased from 0.8 to 2.1 g/dL (p < 0.001), whereas the albumin-to-gamma-globulin ratio decreased from 4.2 to 1.2. Gamma globulins showed the strongest ordinal association (ρ = 0.522; p < 0.001) and moderate single-marker discrimination of seropositivity (area under the receiver operating characteristic curve = 0.768; 95% confidence interval: 0.755–0.780). These findings define the CanL proteinogram as a progressive, quantifiable immunopathological phenotype, dominated by gamma-region expansion and reciprocal albumin decline, supporting serum protein electrophoresis as a reference tool for interpretation and monitoring.

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Serum protein capillary electrophoresis can characterise dysproteinaemia in dogs tested for canine leishmaniosis (CanL) as an integrated clinicopathological phenotype of humoral immune activation. This retrospective laboratory-based study evaluated serum protein electrophoretic patterns in 5544 dogs tested for CanL in Portugal between 2009 and 2026. Anti-Leishmania antibody results were classified into five ordered categories: negative, inconclusive, low positive, positive/high positive and very high positive. Total protein, albumin, alpha-1, alpha-2, beta and gamma fractions, albumin-to-globulin and albumin-to-gamma-globulin ratios were analysed. A separately defined reference subpopulation of 254 clinically healthy seronegative dogs aged ≥ 1 to <4 years was used to establish study-specific non-parametric 95% reference intervals. After excluding inconclusive results, binary analysis included 2454 seronegative and 2910 seropositive dogs. Seropositive dogs had higher median gamma globulins than seronegative dogs (1.5 vs. 0.8 g/dL; p < 0.001), with lower albumin-to-globulin (0.7 vs. 1.0; p < 0.001) and albumin-to-gamma-globulin ratios (1.9 vs. 4.2; p < 0.001). Across the ordinal serological scale, gamma globulins increased from 0.8 to 2.1 g/dL (p < 0.001), whereas the albumin-to-gamma-globulin ratio decreased from 4.2 to 1.2. Gamma globulins showed the strongest ordinal association (ρ = 0.522; p < 0.001) and moderate single-marker discrimination of seropositivity (area under the receiver operating characteristic curve = 0.768; 95% confidence interval: 0.755–0.780). These findings define the CanL proteinogram as a progressive, quantifiable immunopathological phenotype, dominated by gamma-region expansion and reciprocal albumin decline, supporting serum protein electrophoresis as a reference tool for interpretation and monitoring.

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An automated cartridge-based Vcheck M Canine Vector 8 Panel for qualitative detection of Leishmania spp. DNA in canine lymph node aspirates, an off-label specimen type, using laboratory qPCR as the comparator, was evaluated. Fifty-seven residual lymph node aspirate suspensions from dogs investigated for suspected canine leishmaniosis (CanL) were tested. Reference qPCR detected L. infantum DNA in 29 samples. Vcheck M was positive in 22/29 qPCR-positive samples and negative in 28/28 qPCR-negative samples, corresponding to positive percent agreement/sensitivity of 75.9% (95% CI, 56.5–89.7) and negative percent agreement/specificity of 100.0% (95% CI, 87.7–100.0). Agreement was substantial (Cohen’s kappa, 0.76), and discordance was asymmetric (McNemar p = 0.016). Vcheck-negative/qPCR-positive results were mainly observed at low qPCR parasite loads: 6/7 discordant samples contained ≤30 parasites/mL, whereas all samples with ≥500 parasites/mL were Vcheck positive. Among Vcheck-positive clinical samples, Vcheck Ct correlated inversely with log10 qPCR parasite load (Spearman rho = −0.77; p < 0.001). In a single-run dilution series, the lowest instrument-positive L. infantum standard was 103 parasites/mL. Purified L. major, L. braziliensis, and L. tropica DNA were also detected. Vcheck M showed high specificity as a rapid rule-in test for Leishmania spp. detection in canine lymph node aspirates. However, negative results should not exclude infection in symptomatic or strongly suspected dogs and should be confirmed by qPCR, particularly when low parasite burden is plausible or when the result is critical for diagnostic or therapeutic decision-making.

An automated cartridge-based Vcheck M Canine Vector 8 Panel for qualitative detection of Leishmania spp. DNA in canine lymph node aspirates, an off-label specimen type, using laboratory qPCR as the comparator, was evaluated. Fifty-seven residual lymph node aspirate suspensions from dogs investigated for suspected canine leishmaniosis (CanL) were tested. Reference qPCR detected L. infantum DNA in 29 samples. Vcheck M was positive in 22/29 qPCR-positive samples and negative in 28/28 qPCR-negative samples, corresponding to positive percent agreement/sensitivity of 75.9% (95% CI, 56.5–89.7) and negative percent agreement/specificity of 100.0% (95% CI, 87.7–100.0). Agreement was substantial (Cohen’s kappa, 0.76), and discordance was asymmetric (McNemar p = 0.016). Vcheck-negative/qPCR-positive results were mainly observed at low qPCR parasite loads: 6/7 discordant samples contained ≤30 parasites/mL, whereas all samples with ≥500 parasites/mL were Vcheck positive. Among Vcheck-positive clinical samples, Vcheck Ct correlated inversely with log10 qPCR parasite load (Spearman rho = −0.77; p < 0.001). In a single-run dilution series, the lowest instrument-positive L. infantum standard was 103 parasites/mL. Purified L. major, L. braziliensis, and L. tropica DNA were also detected. Vcheck M showed high specificity as a rapid rule-in test for Leishmania spp. detection in canine lymph node aspirates. However, negative results should not exclude infection in symptomatic or strongly suspected dogs and should be confirmed by qPCR, particularly when low parasite burden is plausible or when the result is critical for diagnostic or therapeutic decision-making.