veterinary sciences Article Exploring the Relationship between Neutrophil Activation and Different States of Canine L. infantum Infection: Nitroblue Tetrazolium Test and IFN-γ Carles Blasi-Brugué † , Icíar Martínez-Flórez † , Marta Baxarias , Joan del Rio-Velasco and Laia Solano-Gallego * Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; carles.blasi@uab.cat (C.B.-B.); imflorez.vet@gmail.com (I.M.-F.); martabaxcan@gmail.com (M.B.); rio.joan@gmail.com (J.d.R.-V.) * Correspondence: laia.solano@uab.cat † These authors contributed equally to this work. Simple Summary: This study aimed to understand the role of neutrophils in canine leishmaniosis (CanL) by assessing neutrophil activation and its relationship with different states of Leishmania infantum infection and antibody and IFN-γ production. The results showed that sick dogs in stage I-mild disease had significantly higher neutrophil activation compared to healthy seronegative and seropositive dogs and sick dogs in advanced stages (II, III–IV). Healthy seropositive dogs exhibited higher neutrophil activation compared to all other groups except sick dogs in stage I. Dogs in advanced disease stages (II, III–IV) did not show significant differences in neutrophil activation compared to healthy seronegative dogs. Furthermore, dogs in stage I had significantly higher IFN-γ concentrations compared to healthy seronegative and sick dogs in advanced disease stages. Dogs in stage II showed higher IFN-γ concentrations compared to healthy seronegative dogs, while no significant differences were observed in dogs in stage III–IV. Healthy seropositive dogs had elevated Citation: Blasi-Brugué, C.; IFN-γ concentrations compared to healthy seronegative dogs and dogs in stage III–IV. These findings Martínez-Flórez, I.; Baxarias, M.; del indicate that neutrophil activation is predominant in dogs with mild disease and healthy seropositive Rio-Velasco, J.; Solano-Gallego, L. dogs with an association with potent IFN-γ production. Exploring the Relationship between Neutrophil Activation and Different Abstract: This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing States of Canine L. infantum Infection: neutrophil activation and its relationship with different states of L. infantum infection and antibody Nitroblue Tetrazolium Test and and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy- IFN-γ. Vet. Sci. 2023, 10, 572. seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III–IV https://doi.org/10.3390/ (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neu- vetsci10090572 trophil activation in stage I (median:17.17, range: [7.33–31.50]%) compared to in healthy-seronegative Academic Editors: Anastasia Diakou (4.10 [1.20–18.00]%), healthy-seropositive (7.65 [3.98–21.74]%), stage II (6.50 [1.50–28.70]%), and stage and Donato Traversa III–IV (7.50 [3.00–16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher val- ues than all groups except stage I. Stages II and III–IV did not show significant differences compared Received: 19 July 2023 Revised: 4 September 2023 to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, Accepted: 11 September 2023 range: [0–3998.00] pg/mL) than healthy-seronegative (0 [0–109.50] pg/mL) (p = 0.0002), stage II Published: 13 September 2023 (9.00 [0–5086.00] pg/mL) (p = 0.045), and stage III–IV (3.50 [80.00–548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III–IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs Copyright: © 2023 by the authors. (p = 0.001) and dogs in stage III–IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs Licensee MDPI, Basel, Switzerland. with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation This article is an open access article and the production of IFN-γ was found. distributed under the terms and conditions of the Creative Commons Keywords: NBT; dog; immunity; interferon-gamma; neutrophil; leishmaniosis; oxidative burst; Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ oxidative metabolism 4.0/). Vet. Sci. 2023, 10, 572. https://doi.org/10.3390/vetsci10090572 https://www.mdpi.com/journal/vetsci Vet. Sci. 2023, 10, 572 2 of 20 1. Introduction Canine leishmaniosis (CanL) is a zoonotic vector-borne disease caused by the proto- zoan parasite Leishmania infantum and primarily transmitted through the bite of female Phlebotomine sand flies [1]. It has a worldwide distribution and is considered endemic in regions, such as the Mediterranean basin, Latin America, and southern Asia [1]. Dogs serve as the main reservoir for this infection. Leishmania infantum is an obligate intracellular parasite that infects and survives within various myeloid lineage cells, including mono- cytes, macrophages, dendritic cells, and neutrophils. Its presence in the body has unique implications for the immune system during the course of the infection [2]. Clinical manifestations of CanL can vary widely, ranging from subclinical infection to severe systemic disease [1]. The severity of the disease is classified into four stages: mild disease-LeishVet stage I, characterized by mild clinical signs like papular dermatitis, exhibit- ing no noteworthy clinicopathological abnormalities, and displaying antibody levels that range from negative to low; moderate disease-LeishVet stage II, characterized by diffuse clinical manifestations, like extensive skin lesions, widespread lymph node enlargement or weight loss, clinicopathological abnormalities without a rise in creatinine concentrations, and antibody levels varying from weakly positive to strongly positive; severe disease- LeishVet stage III, defined by extensive clinical manifestations along with clinical signs linked to the deposition of immune complexes (like uveitis or glomerulonephritis), clin- icopathological abnormalities, including either IRIS Stage 1 proteinuric or IRIS Stage 2 chronic kidney disease (CKD), accompanied by antibody levels ranging from moderate to high positivity; and very severe disease-LeishVet stage IV, determined by diffuse clinical signs, clinicopathological abnormalities, and advanced CKD stage 3 or 4, accompanied by antibody levels ranging from moderate to high positivity [1]. The interplay between the host’s innate and adaptive immune responses in response to the parasite infection plays a crucial role in determining the clinical signs and overall outcome of the disease [3,4]. Dogs with a predominant cellular immune response are associated with disease resistance or subclinical forms, while dogs exhibiting a predominantly humoral response are associated with clinical illness [2,5]. Adaptive T-cell immunity has been widely investigated to play a crucial role in disease development and resistance. The T helper 1 (Th1)-mediated immune response, characterized by the secretion of cytokines, such as interferon-gamma (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α), is associated with immune protection [4,6]. Conversely, the Th2-mediated immune response leads to the production of cytokines, like interleukin-4 (IL-4), interleukin-10 (IL-10), and transforming growth factor- beta (TGF-β), which are linked to a predominantly humoral immune response and disease progression [4,6]. A balanced response between proinflammatory Th1 CD4+ T cells that inhibit parasite replication and an immune regulatory response mediated by T regulatory 1 (Tr1) cells is crucial for disease control [2]. The former stimulates macrophage activity through IFN-γ production, promoting parasite elimination [7]. However, prolonged ex- posure to antigens can lead to T cell exhaustion, resulting in reduced T cell proliferation and IFN-γ production. This, in turn, allows for parasite survival and expansion within macrophages [7]. In recent years, growing evidence has highlighted the significant role of the innate immune response in controlling Leishmania infection. Among the components of the innate immune system, neutrophils have emerged as key players, particularly in the early stages of Leishmania infection [8]. While macrophages are considered the primary host target cells for the replication of L. infantum, neutrophils also play a distinct role in the control or progression of leishmaniosis, thanks to their ability to kill parasites and produce interleukins [9,10]. As the first line of defense, neutrophils are the initial cells that migrate to the site of infection and internalize the Leishmania promastigote [11–14]. Upon phagocytosis, neutrophils activate their defense mechanisms, including the production of reactive oxygen species (ROS), which is a crucial antileishmanial mechanism produced in phagocytes (oxidative burst) [15–18]. The phagocyte’s NADPH oxidase generates the superoxide anion (O2 − ), which serves as a precursor to hydrogen peroxide and other ROS [15–17,19–21]. Vet. Sci. 2023, 10, 572 3 of 20 Subsequently, the neutrophil releases these ROS within the phagosomes containing L. infantum amastigotes, effectively killing the parasite [19]. In addition to the oxidative burst, neutrophils that ingest Leishmania amastigotes initiate further defensive strategies. These include the secretion of TNF-α and IFN-γ, which aid in stimulating macrophage activation and recruitment to the infection site [22,23] and the release of neutrophil extracellular traps, which also potentially play a significant role in combatting protozoal infections, such as Leishmania [24]. However, Leishmania amastigotes have the ability to deactivate the oxidative activity of polymorphonuclear leukocytes, allowing them to survive within neutrophils and exploit them as a means to hide from the immune system while silently infecting the final host cell, the macrophage [10,22,25]. Alternatively, if the oxidative burst is successful and limits parasite growth within cells, the infection can be controlled [26,27]. Therefore, the ability or predisposition of neutrophils to activate the oxidative burst can have a significant impact on disease progression or containment. Interferons (IFNs) are a family of proteins that exhibit strong antiviral and antibacterial effects and play crucial roles in regulating effector cells within both innate and adaptive immune responses [28–31]. Among these, IFN-γ stands out for having the most diverse and potent effects [32]. While much of the research on IFN-γ has focused on its effects on adaptive immunity, recent studies have revealed its ability to modulate myeloid cell activity, including neutrophils, in diverse ways. These include enhancing NADPH oxidase activity and the oxidative burst, promoting nitric oxide (NO) production, increasing the expression of MHCII proteins, altering cytokine and chemokine production, inhibiting chemotaxis, and suppressing apoptosis [33]. Although these effects have been predominantly observed in vitro based on mature neutrophils, studies suggest that more substantial changes occur when IFN-γ interacts with maturing myeloid cells [34]. Thus, IFN-γ could potentially have an impact on the capacity of neutrophils to eliminate phagocyted Leishmania amastigotes and promastigotes by stimulating the cellular immune response and activating phagosomes to induce an oxidative burst. Therefore, a positive relationship within IFN-γ and the oxidative metabolism of neutrophils could be suspected. The nitroblue tetrazolium reduction test (NBT) is a diagnostic assay that utilizes the ability of neutrophils and macrophages to generate free radicals (oxidative burst) during the process of phagocytosis [35,36]. This test relies on the reduction of nitroblue tetrazolium, a colorless compound, to formazan, a blue compound, by the NADPH oxidase present in activated neutrophils within the phagocytic vacuole. As a result, the cytoplasm of the cells undergoes a color change, indicating the production of ROS [36]. The NBT reaction provides an assessment of the ROS-generating activity in the cytoplasm of cells and has been shown to correlate strongly with the levels of ROS produced during the oxidative burst [37,38]. By employing conventional light microscopy, the rate of NBT can be determined by calculating the percentage of neutrophils with formazan present in their cytoplasm, reflecting the activation status of neutrophils and monocytes in the peripheral blood [35]. The primary objective of this study was to assess the variations in peripheral blood neutrophil ROS generation in dogs with different stages of L. infantum infection by means of the NBT. Additionally, we aimed to investigate the potential association between neutrophil ROS generation and IFN-γ concentrations, as well as antibody levels, in dogs with different states of L. infantum infection. 2. Materials and Methods All dogs included in this study belonged to volunteer owners who had granted permission to a physical examination and blood collection from their dogs through a signed consent form. 2.1. Dogs and Clinical Data Dogs of different breeds from an endemic area were selected to enter the study. A complete blood count, serum biochemistry profile (at least containing creatinine, alanine Vet. Sci. 2023, 10, 572 4 of 20 aminotransferase, and albumin), urinalysis, serum electrophoresis, and ELISA serology were performed for all dogs, as well as cytology of the lesions in dogs in stage I. A total of 128 dogs were enrolled in this study and divided into five groups according to the LeishVet staging system [1]: Group 1 included healthy seronegative dogs with no clinical signs or hematological or biochemical abnormalities (n = 25); Group 2 included Leishmania-seropositive but clinically healthy dogs with no clinical signs, hematological and serum and urinary biochemical parameters within normal limits, and presenting low to medium antibody levels (n = 21); Group 3 included dogs with mild disease-LeishVet stage I presenting papular dermatitis as the sole clinical sign [39], the identification of intrale- sional amastigotes via cytology, hematological, serum, and urinary biochemical parameters within normal limits (including creatinine < 1.4 mg/dL), and presenting negative to low antibody levels (n = 25); Group 4 included dogs with moderate disease-LeishVet stage II, including the characteristic clinical signs (diffuse clinical manifestations, like extensive skin lesions, widespread lymph node enlargement, or weight loss among others), hematological and serum biochemical abnormalities with normal creatinine values (<1.4 mg/dL), and medium-to-high antibody levels (n = 41); Group 5 included dogs with severe disease- LeishVet stage III and very severe disease-Leishvet stage IV, including the characteristic clinical signs, hematological, biochemical abnormalities (including creatinine values of 1.4–2.8 mg/dL or UPC > 1 for stage III and creatinine values > 2.8 for stage IV or UPC > 5), and presenting positive antibody levels (n = 16) [1]. The urine protein creatinine ratio was assessed in 12/25 dogs in stage I and in all dogs in stages II, III, and IV. Leishmania infantum- specific antibody levels were assessed in all dogs using an end point ELISA. As for IFN-γ, this test was conducted on most of the dogs, although technical limitations prevented its performance in all cases. IFN-γ measurements were obtained from 10 out of 25 healthy seronegative dogs, 15 out of 21 healthy seropositive dogs, 24 out of 25 stage I dogs, 34 out of 41 stage II dogs, and 13 out of 16 stage III/IV dogs. A follow-up of up to one year was available for all dogs with papular dermatitis without conventional anti-Leishmania systemic treatment instituted. Some dogs in this group received local treatment whit either topical meglumine antimoniate as a lotion spray (n = 6/25), polyhexamethylene biguanide alone or in combination with Toll like receptor 4 agonist as a spray (n = 9/25), or placebo (n = 9/25), and one received no treatment at all [39]. 2.2. Blood Collection The blood was collected via jugular or cephalic venipuncture and placed in EDTA tubes for the hematologic analysis and the NBT test, into heparin tubes to perform cytokine release whole blood assay, and into serum tubes for the antileishmanial antibody quantification. 2.3. Antileishmanial Antibody Quantification via ELISA Antibody levels against L. infantum were assessed using an in-house Enzyme-Linked ImmunoSorbent Assay (ELISA), as outlined in previous studies [3,40]. Dog sera were diluted to a ratio of 1:800 in a phosphate buffer solution (PBS) containing Tween 20 and 1% dry milk. This mixture was then incubated in plates previously coated overnight with sonicated promastigotes of L. infantum (MHOM/MON-1/LEM-75) obtained from an infected dog at a concentration of 20 µg/mL, and the incubation took place at 37 ◦ C for 1 h [41]. Following this, the plates underwent a series of washes using PBS-Tween and PBS. Next, Protein A conjugated to horseradish peroxidase (Peroxidase Conjugate Protein A; Merck KGaA, Darmstadt, Germany) was added to the plates at a dilution of 1:30,000 and incubated for 1 h at 37 ◦ C. After another round of washing, the plates were treated with o-phenylenediamine and substrate buffer (SIGMAFAST OPD; Merck KGaA, Darmstadt, Germany). The reaction was terminated using 5 M H2 SO4 . Absorbance readings were taken at 492 nm using a spectrophotometer (MB-580 HEALES; Shenzhen Huisong Technology Development Co., Ltd., Shenzhen, China). The quantification of results was performed in terms of ELISA units (EUs), normalized against positive canine serum utilized as a calibrator and set at 100 EU. Vet. Sci. 2023, 10, 572 5 of 20 The threshold was established at 35 EU [39]. Serum samples were categorized as negative when their value fell below 35 EU. If the result ranged from 35 EU up to but not exceeding 150 EU, it was considered low positive. Similarly, if the result fell between 150 EU and 300 EU, it was categorized as medium positive. Finally, if the value equaled or surpassed 300 EU, it was classified as high positive. For a more in-depth examination of samples identified as medium or high positive, an ELISA involving a two-fold serial dilution was conducted. This process was initiated at a dilution of 1:800 and continued with an additional 7 to 11 dilutions. Quantification was predicated on arbitrary units (EU) in relation to a calibrator set at 100 EU, which corresponded to an optical density (OD) value of 1 at the 1:800 dilution. The mean values of dilutions displaying an OD approximate to one were chosen for calculating the EU. This computation was executed using the following formula: (sample OD/calibrator OD) × 100 × dilution factor [40]. 2.4. Nitroblue Tetrazolium Reduction Test For each EDTA tube, blood was drawn and filled into three hematocrit capillary microtubes (Servoprax, Wiesel, Germany). These microtubes were then subjected to cen- trifugation at 2910× g for 5 min (Fugevet+ GDC005, Nahita International Ltd., London, UK) to isolate the buffy coat. The obtained buffy coat was transferred to an Eppendorf tube, where it was mixed with an equal volume of 0.1% NBT solution (1:1) (N6876, Sigma-Aldrich Co., St. Louis, MO, USA). The mixture was gently agitated and then placed in a heater at 37.5 ◦ C for a duration of 15 min (INB 200, Memmert GmbH + Co. KG, Schwabach, Germany), followed by an additional 15 min at room temperature. Subsequently, two blood smears were prepared from each Eppendorf tube, with 3 µL of NBT-stained blood being placed on each glass slide. These slides were further stained using the Diff-Quik method. To assess the NBT rate, a total of 300 neutrophils displaying a clear morphology were counted on each slide using standard light microscopy. Neutrophils that were aggregated or damaged were excluded from the count. The NBT rate was calculated by determining the percentage of activated neutrophils, characterized by the presence of blue-black formazan deposits, among the total counted neutrophils [42]. Dogs were considered to have an increased NBT rate when the NBT value exceeded the cut-off. This cut-off was calculated by considering the NBT values of the 25 healthy seronegative dogs as the control group. A high sensitivity was desired for this cut-off. The standard deviation was added to the mean value of this group, resulting in a cut-off of 10.80%. With this cut-off, considering the healthy seronegative dogs as negative and the dogs with papular dermatitis as positive, the sensitivity was 92% and the specificity 84%. 2.5. Cytokine Release Whole Blood Assay and Determination of Canine IFN-γ The heparinized cytokine release whole blood assay was conducted following estab- lished procedures [3]. To outline the process, the assay involved incubating whole blood separately under three distinct conditions: (i) medium alone (unstimulated); (ii) medium containing L. infantum soluble antigen (LSA) at a concentration of 10 µg/mL [43]; and (iii) medium supplemented with the mitogen concanavalin A (ConA, 100 mg, Medicago® , Uppsala, Sweden) at a concentration of 10 µg/mL. LSA was obtained through a process involving triple cycles of freezing and thawing of cultured L. infantum promastigotes (MHOM/MON-1/LEM-75) suspended at a concentration of 1 × 109 cells/mL in PBS. Sub- sequently, the supernatant was harvested following centrifugation (8.000× g, 20 min, 4 ◦ C), adhering closely to the methodology outlined in Carrillo et al., 2015, albeit with minor adjustments [44]. Following five days of incubation at 37 ◦ C in an environment containing 5% CO2 , supernatants were obtained through centrifugation at 300× g for a duration of 10 min. These supernatants were then collected and stored at −80 ◦ C until the testing phase. The measurement of IFN-γ was carried out for all samples utilizing a commercially available sandwich ELISA (Du-oSet® ELISA by Development System R&DTM, Abingdon, UK) using a canine IFN-γ antibody. A standard curve for canine IFN-γ was established Vet. Sci. 2023, 10, 572 6 of 20 through a computer-generated four-parameter logistic curve-fit using the MyAssays pro- gram (http://www.myassays.com/) [3]. Dogs were categorized as IFN-γ producers if the concentration of L. infantum-specific IFN-γ exceeded or was equal to 110 pg/mL after accounting for the medium-alone baseline [43]. 2.6. Statistical Analysis The statistical analysis of data was performed using GraphPad Prism 8.0.1 for Win- dows software (GraphPad Software, San Diego, CA, USA). Several normality tests (Anderson–Darling, D’Agostino and Pearson, Shapiro–Wilk, and Kolmogorov–Smirnov) were performed to assess the normality of the different vari- ables (NBT rate, serological results, and IFN-γ concentration). No variables followed a normal distribution except for the NBT rate in stage I and stage III–IV considering a p-value < 0.05 as statistically significant. Therefore, non-parametric tests were used to assess the data, except for the unpaired t-test (parametric test) performed between the parameters with a normal distribution. A non-parametric Mann–Whitney U-test was used for quantitative variables to compare two groups. A Kruskal–Wallis test and a Dunn’s multiple comparisons test were used to compare more than two groups when data did not follow a normal distribution. If data followed a normal distribution, an ordinary one-way ANOVA and a Tukey’s multiple comparisons test were performed. The Chi-square test or Fisher’s exact test was performed to determine if an association between categorical variables existed. The Spearman (non-normal distribution) or Pearson (normal distribution) correlation coefficient was used to evaluate if there were correlations among different parameters studied. Results were considered statistically significant at a p-value < 0.05. 3. Results 3.1. Dog Clinical Characteristics and Performed Tests Dog clinical characteristics and results of performed tests are summarized and dis- played in Tables 1 and 2. Table 1. Distribution of qualitative characteristics including breed, sex, positive/negative NBT, positive/negative serology, and productive/non-productive status of IFN-γ among dogs categorized within the five states of infection studied. Healthy Healthy Stage I Stage II Stage III–IV p-Value Qualitative Characteristics Seronegative Seropositive (n = 25) (n = 41) (n = 16) (Chi-Square, df ) (n = 25) (n = 21) Crossbred (n = 54) 9 (36.00%) 11 (52.38%) 14 (56.00%) 16 (39.02%) 4 (25.00%) 0.25 Breed (n = 128) Purebred (n = 74) 16 (64.00%) 10 (47.62%) 11 (44.00%) 25 (60.98%) 12 (75.00%) (5.30, 4) Male (n = 74) 12 (48.00%) 10 (47.62%) 16 (64.00%) 23 (56.10%) 13 (81.25%) 0.2 Sex (n = 128) Female (n = 54) 13 (52.00%) 11 (52.38%) 9 (36.00%) 18 (43.90%) 3 (18.75%) (5.90, 4) Increased (n = 46) 4 (16.00%) 5 (23.81%) 23 (92.00%) 10 (24.39%) 3 (18.75%) <0.0001 * NBT (n = 128) WNL (n = 82) 21 (84.00%) 16 (76.19%) 2 (8.00%) 31 (75.61%) 13 (81.25%) (44.60, 4) Serology Positive (n = 84) 0 (0.00%) 21 (100.00%) 6 (24.00%) 41 (100.00%) 16 (100.00%) <0.0001 * (n = 128) Negative (n = 44) 25 (100.00%) 0 (0.00%) 19 (76.00%) 0 (0.00%) 0 (0.00%) (107.80, 4) Producers (n = 34) 0 (0.00%) 9 (60.00%) 12 (50.00%) 9 (26.47%) 4 (30.77%) 0.01 * IFN-γ (n = 97) Non-prod. (n = 63) 10 (100.00%) 6 (40.00%) 12 (50.00%) 25 (73.53%) 9 (69.23%) (12.54, 4) NBT = nitroblue tetrazolium reduction test, WNL = within normal limits, IFN-γ = interferon-gamma, Non-prod. = non-producers, ELISA = Enzyme-Linked ImmunoSorbent Assay. * Significant differences were observed between groups. Vet. Sci. 2023, 10, 572 7 of 20 Table 2. Median, minimum, and maximum values for quantitative traits, like age, NBT, serology, and IFN-γ within each group. Quantitative Healthy Healthy p-Value Stage III–IV Characteristics Seronegative Seropositive Stage I (n = 25) Stage II (n = 41) (Kruskal-Wallis (n = 16) Median (Min–Max) (n = 25) (n = 21) Test) Age (years) 1.30 (1.00–1.60) 5.00 (1.00–13.00) 0.50 (0.25–7.00) 4.00 (1.00–13.00) 7.50 (1.00–11.00) <0.0001 * NBT (%) 4.13 (1.15–17.96) 7.65 (3.98–21.74) 17.17 (7.33–31.50) 6.50 (1.50–28.70) 7.50 (3.00–16.75) <0.0001 * IFN-γ (pg/mL) 0 (0–109.50) 204.10 (0–4763.00) 127.90 (0–3998.00) 9.00 (0–5086.00) 3.48 (0–548.80) 0.001 * Serology (ELISA 133.90 1894.00 1857.00 5.50 (0.30–28.49) 21.42 (2.76–227.10) <0.0001 * units) (75.79–956.30) (57.16–10,293.00) (96.79–11,114.00) NBT = nitroblue tetrazolium reduction test, IFN-γ = interferon-gamma, ELISA = Enzyme-Linked ImmunoSorbent Assay. * Significant differences were observed between groups. Forty-two percent of dogs were crossbred (n = 54), and the other most represented breeds were Beagle (n = 10), German shepherd (n = 8), and Greyhound (n = 5). 3.2. Age-Stratified Analysis of NBT Rate in Dogs with Papular Dermatitis (Stage I): Insights from Younger and Older Canine Cohorts Dogs within the stage I group were further subdivided between dogs younger than one year of age (n = 22) and dogs older than one year of age (n = 3) for a further statistical analysis. No statistical differences were found when comparing the NBT rate of dogs in both groups (p = 0.674). Additionally, the statistical study comparing the stage I group with the other groups was repeated but only taking in consideration the dogs that were older than 1 year of age, showing similar results to the previous study performed. Stage I (older than 1 year of age) had a significantly higher NBT rate than dogs in the healthy seronegative group (p = 0.02), the healthy seropositive group (p = 0.011), the stage II group (p = 0.002), and the stage III–IV group (p < 0.0001). 3.3. Follow-Up of Dogs with Papular Dermatitis (Stage I) Over the following months after diagnosis, the majority of the dogs exhibited a notable improvement or the resolution of their cutaneous lesions, and the antibody levels demonstrated a declining trend. However, among the 25 dogs studied, three exhibited a deterioration in their cutaneous lesions, which progressed to the point of ulceration in two cases. Consequently, a systemic conventional anti-Leishmania treatment approach was initiated to address their condition. For the purpose of conducting a statistical analysis, the three dogs that experienced a worsening of their clinical signs were categorized as belonging to group PD-B, and the remaining 22 dogs that displayed an improvement or the resolution of their clinical signs without the need for systemic treatment were designated as members of group PD-A. Detailed results pertaining to NBT, IFN-γ, and serology, both for these delineated groups and for each individual dog within group PD-B, can be found in Table 3. Upon conducting a comparative analysis of the NBT, IFN-γ, and serology results between the two groups, no statistically significant differences were found (p = 0.074, p = 0.176, and p = 0.271, respectively). However, it is worth noting that the sample size for group PD-B was limited to only three dogs, which could have influenced the robustness of the statistical outcomes. Interestingly, despite the limitations in sample size, a significant correlation was estab- lished between the number of dogs surpassing the NBT cut-off value and the achievement of a clinical improvement without the requirement for systemic treatment. This observation indicated that dogs in group PD-B (those experiencing worsening clinical signs) were more prone to exhibit NBT results within the normal range, unlike dogs in group PD-A (demonstrating an improvement without systemic treatment). However, no corresponding link was identified between the various groups and the status of being an IFN-γ producer or having a positive serology. Vet. Sci. 2023, 10, 572 8 of 20 Table 3. NBT and IFN-γ results for dogs that presented with papular dermatitis and divided into two groups: Group PD-A (clinical improvement without systemic treatment) and Group PD-B (clinical worsening and need for systemic treatment). NBT Status Serological NBT (%) IFN-γ IFN-γ Status Serology Clinical Evolution (Cut-Off = 10.8) Status Median: 17.59 Median: 171.90 Median: 18.21 Group PD-A 22/22 Over Partial or total (min: 9.67, (min: 0, 10/10 Producers (min: 2.76, 5/22 positive (n = 22) cutoff improvement max: 31.50) max: 3998.00) max: 227.10) Median: 8.80 Median: 7.03 Median: 27.41 Group PD-B (min: 7.33, 2/3 WNL (min: 0, 1/3 Producer (min: 22.34, 1/3 positive - (n = 3) max: 20.83) max: 234.20) max: 79.90) Worsening of cutaneous clinical Dog PD-B.1 8.80 WNL 7.03 Non-producer 79.90 Positive signs: diffuse worsening. Worsening of cutaneous clinical Dog PD-B.2 7.33 WNL 234.24 Producer 27.41 Negative signs: ulcerative lesion at the nose bridge. Worsening of cutaneous clinical signs: ulcerative Dog PD-B.3 20.83 Over cutoff 0 Non-producer 22.34 Negative lesion at the level of the right lower eyelid. Group PD-A = dogs diagnosed with papular dermatitis that showed an improvement in clinical signs without systemic treatment, Group PD-B = dogs diagnosed with papular dermatitis that showed the worsening of clinical signs and in which systemic treatment was considered necessary, NBT = nitroblue tetrazolium reduction test, WNL = within normal limits, IFN-γ = interferon-gamma. 3.4. Total Leucocyte Concentration and Differential Leukocyte Concentration The results from the total leucocyte concentration and the differential leukocyte con- centration analysis within the different groups studied are displayed in Table 4. Upon scrutinizing the outcomes across different groups, no statistically significant differences were identified in terms of total leukocyte, neutrophil, and eosinophil concentrations. How- ever, significant differences were observed in relation to the lymphocyte concentration. Specifically, healthy seronegative dogs exhibited notably higher lymphocyte concentrations in comparison to the healthy seropositive dogs (p = 0.028), as well as to dogs in stage II (p = 0.048) and stage III–IV (p = 0.001). Interestingly, no substantial variance was found when comparing the lymphocyte concentration between healthy seronegative dogs and dogs in the stage I group. Furthermore, regarding the lymphocyte concentration within the stage I group, significantly higher values were also recorded in contrast to healthy seropositive dogs (p = 0.022), dogs in stage II (p = 0.033), and dogs in stage III–IV (p = 0.001). Notably, no significant differences were evident when comparing the stage II group with the stage III–IV group. Regarding the monocyte concentration, statistically significant differences were ob- served between healthy seropositive dogs and those in stage I (p = 0.004), as well as between dogs in stage II and those in stage I (p = 0.04). In both cases, the stage I group exhibited markedly higher values. When exploring potential correlations between the NBT rate and both neutrophil and lymphocyte concentrations, a comprehensive analysis encompassing all of the dogs collectively revealed no significant correlations (p = 0.491 and p = 0.199, respectively). This pattern persisted when conducting separate evaluations within distinct groups, which included healthy seronegative (p = 0.682 and p = 0.811, respectively), healthy seroposi- tive (p = 0.766 and p = 0.144, respectively), stage I (p = 0.187 and p = 0.507, respectively), stage II (p = 0.408 and p = 0.494, respectively), and stage III–IV (p = 0.625 and p = 0.525, respectively) groups. Vet. Sci. 2023, 10, 572 9 of 20 Table 4. Median (min–max) results for total leukocyte concentrations and differential leukocyte concentrations in each group. Median Healthy Healthy Stage I Stage II Stage III–IV p-Value (Min–Max) Seronegative Seropositive Leucocytes 11,330 (6390–15,020) 9710 (3360–16,920) 11,180 (6200–20,540) 10,470 (3990–19,430) 9650 (3420–13,630) 0.115 (cell/µL) Neutrophils 6675 (3706–9913) 5889 (2755–11,844) 6939 (3300–17,860) 7171 (3032–14,378) 6482 (2633–10,223) 0.936 (cell/µL) Lymphocytes 3006 (1968–3930) 1711 (235–3849) 3590 (900–5800) 1921 (518–4696) 1472 (616–2385) <0.0001 * (cell/µL) Eosinophils 409 (300–934) 514 (63–2369) 500 (20–1100) 479 (0–2176) 386 (0–1204) 0.541 (cell/µL) Monocytes 668 (313–1502) 413 (34–1157) 750 (100–1310) 517 (129–1269) 560 (171–1204) 0.003 * (cell/µL) * Significant differences were observed between groups. Regarding the monocyte concentration, a significant negative correlation with the NBT was observed exclusively within the stage I group (p = 0.005). In contrast, no such correla- tions emerged when examining the healthy seronegative (p = 0.785), healthy seropositive (p = 0.128), stage II (p = 0.451), and stage III–IV (p = 0.73) groups separately. Similarly, no overall correlation was identified when considering all dogs collectively (p = 0.159). Vet. Sci. 2023, 10, 572 10 of 21 3.5. Nitroblue Tetrazolium Reduction Test The results of NBT are depicted in Tables 1 and 2 and Figure 1. A significantly (23.8%)median lower NBT rate (p < 0.0001), was stage II observed in