**Abstract**

Visceral leishmaniasis (VL) is a zoonotic parasitic disease caused by *Leishmania infantum (L. chagasi)* that infects cells of the monocyte-phagocyte system. This work aims to describe the bone marrow parasitism in dogs naturally infected by *L. chagasi*, and to correlate with serum concentrations of cytokines and antibody level. It evaluated 42 dogs, 21 uninfected and 21 infected by *L. infantum*, of both sexes and of different ages; dogs were classified into three clinical stages: stage I, mild disease; stage II, moderate disease; and stage III, severe disease. Parasitic index was determined by real-time polymerase chain reaction (PCR) and cytokine serum concentration by flow cytometry. The average parasitic index of infected dogs was 4.59 × 1010 copies/μl. IL-4 and TNF-α concentrations were higher in infected dogs than in the control group. Antibody levels were positively correlated with IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. Antibody levels were positively correlated with IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. However, this cytokine can be used as a marker to distinguish between different clinical stages.

**Keywords:** *Leishmania infantum*, dogs, cytokines, parasitic index, cytometry

## **1. Introduction**

Visceral leishmaniasis (VL) is a parasitic zoonotic disease caused by the protozoan *Leishmania infantum* (syn. *L. chagasi)*, an intracellular parasite of the phagocytic mononuclear system [1, 2]. In Brazil, VL is transmitted by sandflies, *Lutzomyia longipalpis* [1, 3, 4].

In a global scenario, it is estimated that 300,000 new cases of VL occur with a rate of 20,000 deaths each year, with 94% new cases reported in Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan [5]. While in Latin America, LV spreads from Mexico to Argentina, with the largest number of cases concentrated in Brazil [6]. With the urbanization of VL in Brazil, annually, the country records approximately 3500 new cases, mainly in medium and large cities; probably, it is due to the disordered anthropic occupation of the geographic space [7].

Despite scientific advances, cases of VL are expanding, which has a major impact on public health, as dogs are the main reservoirs in the urban environment and therefore play an important role in the transmission cycle [8, 9].

Canine visceral leishmaniasis (CVL) is characterized by a broad clinical spectrum, from mild and moderate to fatal clinical manifestations. Major clinical signs in dogs include hepatosplenomegaly, lymphadenopathy, exfoliative dermatitis, alopecia, onychogryphosis, keratoconjunctivitis, apathy, anorexia, and severe weight loss [10–13].

The clinical manifestation of CVL depends on the interaction of the parasite with the host immune response [2]. In susceptible dogs, clinicopathological abnormalities are preceded by an evident humoral response and depression of the cellular response, mediated by a non-protective Th2 immune response associated with cytokines IL-4, IL-5, IL-6, and IL-10 [14, 15]. Dogs that do not develop the disease have a protective cellular response (Th1) [16, 17], related to INF-γ, TNF-α, IL-2, and IL-12 cytokines.

Different procedures are used for the diagnosis of CVL [18]. The Brazilian Ministry of Health recommends serology in the investigation of canine disease by the Dual-Path Platform (DPP®) rapid method as a screening test and ELISA as confirmatory test [19]. Other tests are used to demonstrate infection, such as cytology, histopathology [20], and real-time PCR (RT-PCR) [21].

Similarly, determination of parasitic index has become important for early detection, but also evaluation of treatment efficacy and monitoring of relapses [22]. Thus, the aim of this study was to associate parasitic index to serum cytokine concentration in dogs naturally infected by *L. infantum* at different clinical stages of infection.

### **2. Methodological aspects**

The procedures were previously approved by the Ethics Committee on the Use of Animals (ECUA)/UFMT, Brazil (n° 23108.019567/14-1), and collection of clinical samples was authorized by the dog owners by signing the informed consent form.

#### **2.1 Animals**

This study was conducted over a 16-month period, evaluating 42 male and female dogs of different ages and breeds from Barra do Garças, Mato Grosso State, Brazil (latitude, −15.893; longitude, 52.2599; south,15° 53′ 35″; west 52° 15′ 36″). Dogs with canine visceral leishmaniasis (n = 21) were classified into clinical stages at diagnosis as described by Solano et al. [23] and confirmed using the Dual-Path Platform Rapid Test (RT DPP®) and polymerase chain reaction (PCR). A control group (n = 21) was also formed, comprising dogs with no clinical changes and negative results for RT DPP® and conventional PCR.

#### **2.2 Blood and bone marrow sample**

Blood samples (5 mL) were collected by cephalic or jugular venipuncture, placed in tubes without anticoagulant to obtain serum. Serum was obtained by centrifuging the blood sample at 300× *g* for 5 minutes and was then transferred to 2 mL microtubes and stored at −80°C for cytokine dosing.

After dog restraint and local anesthesia with 2% lidocaine, bone marrow samples were obtained from the sternal manubrium, placed in microtubes with 0.5 mL 0.9% sterile NaCl solution, and stored at −20°C for subsequent molecular techniques.

**105**

(UV, 300 nm).

software version 5.0.

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis*

The immunochromatographic rapid test for detection of anti-*Leishmania infantum* antibodies (DPP®—Canine Visceral Leishmaniasis-Bio-Manguinhos/ FIOCRUZ, Rio de Janeiro, Brazil) that uses the recombinant protein K39 (rK39) as an antigen, a cloned 39 amino acid sequence of the specific *L. infantum* kinase

DNA extraction from blood samples was performed by the phenol-chloroform method. The polymerase chain reaction assay was performed using the primers RV1 (sense) 5′-CTT TTC TGG TCC CGC GGG TAG G-3′ and RV2 (antisense) 5′-CCA CCT GGC TAT TTT ACA CCA-3′ [24], which amplifies the DNA fragment of a 145 bp region of conserved kDNA present in *L. infantum*. Amplification used 200 mM dNTP, 1 pM from each primer, a buffer solution (10 mM Tris–HCl and 50 mM KCl, pH 8.3), 2 mM MgCl2, 1.5 U Taq DNA polymerase, and 2 μl of the DNA sample in the final volume of 25 μl. Assays were performed for one cycle at 94°C for 4 minutes, followed by 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and final extension of one cycle at 72°C for 10 minutes. The amplification product was fractionated by 2.0% agarose gel electrophoresis, stained with red gel spot, and visualized on a transilluminator

Quantitative PCR (qPCR) was performed in triplicate using the StepOne™ Real-Time PCR System Sequence Detection System (Applied Biosystems) targeting RV1–5′-CTT TTC TGG TCC GGG TAG G-3′ primers and RV2–5′-CCA CCT GGC TAT TTT ACA CCA-3′ amplifying a 145 bp sequence of *L. infantum-*specific kDNA [24]. Reactions were prepared in a 25 μl final volume containing SYBR Green Master Mix, 0.3 μM of each primer, and 2 μl of target DNA. Amplification conditions included an initial incubation step at 94°C for 10 minutes, followed by 40 cycles of amplification, 94°C for 15 seconds, and 60°C for 60 seconds. The standard curve was established for each assay using known amounts of TOPO PCR 2.1 plasmid (Invitrogen Corp.) containing *L. infantum* kDNA gene. Serial (10×) dilutions of

Serum cytokine concentration (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was assessed using the Cytometric Bead Array (CBA) Kit (BD Bioscience, USA) and evaluated by a flow cytometer (FACSCalibur®, BD Bioscience, USA). The reading was done using the CellQuest. Data were analyzed in FCAP array

Immunoglobulin concentrations (IgM and IgG) in the sera were determined by turbidimetric method. For 1:11 (v/v) IgM and 1:15 (v/v) IgG, antibody concentrations were determined using IgM (Bioclin®, Brazil, Ref K063) and IgG (Bioclin®, Brazil, Ref K062) antiserum diluted with 1:12 (v/v). The calibration curve obtained from the Multical calibrator (Bioclin®, Brazil, Ref K064) was used to determine the standard curve for each immunoglobulin. Positive and negative serum samples, standards, and controls were placed in 500 μl buffer solution (0.15 mol/L sodium

–2.9×108

copies of the plasmid were

*DOI: http://dx.doi.org/10.5772/intechopen.90573*

**2.3 Immunochromatographic rapid test: RT DPP® kit**

**2.4 DNA extraction, conventional PCR, and qPCR**

the recombinant plasmid containing 2.9×104

performed and used on the standard curve.

**2.5 Cytokine quantification by flow cytometry**

**2.6 Determination of serum immunoglobulins**

region, was performed according to the manufacturer's guidance.

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.90573*

#### **2.3 Immunochromatographic rapid test: RT DPP® kit**

*Parasitology and Microbiology Research*

weight loss [10–13].

and IL-12 cytokines.

infection.

**2.1 Animals**

**2. Methodological aspects**

tive results for RT DPP® and conventional PCR.

microtubes and stored at −80°C for cytokine dosing.

**2.2 Blood and bone marrow sample**

Despite scientific advances, cases of VL are expanding, which has a major impact on public health, as dogs are the main reservoirs in the urban environment and there-

Canine visceral leishmaniasis (CVL) is characterized by a broad clinical spectrum, from mild and moderate to fatal clinical manifestations. Major clinical signs in dogs include hepatosplenomegaly, lymphadenopathy, exfoliative dermatitis, alopecia, onychogryphosis, keratoconjunctivitis, apathy, anorexia, and severe

The clinical manifestation of CVL depends on the interaction of the parasite with the host immune response [2]. In susceptible dogs, clinicopathological abnormalities are preceded by an evident humoral response and depression of the cellular response, mediated by a non-protective Th2 immune response associated with cytokines IL-4, IL-5, IL-6, and IL-10 [14, 15]. Dogs that do not develop the disease have a protective cellular response (Th1) [16, 17], related to INF-γ, TNF-α, IL-2,

Different procedures are used for the diagnosis of CVL [18]. The Brazilian Ministry of Health recommends serology in the investigation of canine disease by the Dual-Path Platform (DPP®) rapid method as a screening test and ELISA as confirmatory test [19]. Other tests are used to demonstrate infection, such as cytol-

Similarly, determination of parasitic index has become important for early detection, but also evaluation of treatment efficacy and monitoring of relapses [22]. Thus, the aim of this study was to associate parasitic index to serum cytokine concentration in dogs naturally infected by *L. infantum* at different clinical stages of

The procedures were previously approved by the Ethics Committee on the Use of Animals (ECUA)/UFMT, Brazil (n° 23108.019567/14-1), and collection of clinical samples was authorized by the dog owners by signing the informed consent form.

This study was conducted over a 16-month period, evaluating 42 male and female dogs of different ages and breeds from Barra do Garças, Mato Grosso State, Brazil (latitude, −15.893; longitude, 52.2599; south,15° 53′ 35″; west 52° 15′ 36″). Dogs with canine visceral leishmaniasis (n = 21) were classified into clinical stages at diagnosis as described by Solano et al. [23] and confirmed using the Dual-Path Platform Rapid Test (RT DPP®) and polymerase chain reaction (PCR). A control group (n = 21) was also formed, comprising dogs with no clinical changes and nega-

Blood samples (5 mL) were collected by cephalic or jugular venipuncture, placed in tubes without anticoagulant to obtain serum. Serum was obtained by centrifuging the blood sample at 300× *g* for 5 minutes and was then transferred to 2 mL

After dog restraint and local anesthesia with 2% lidocaine, bone marrow samples were obtained from the sternal manubrium, placed in microtubes with 0.5 mL 0.9% sterile NaCl solution, and stored at −20°C for subsequent molecular techniques.

fore play an important role in the transmission cycle [8, 9].

ogy, histopathology [20], and real-time PCR (RT-PCR) [21].

**104**

The immunochromatographic rapid test for detection of anti-*Leishmania infantum* antibodies (DPP®—Canine Visceral Leishmaniasis-Bio-Manguinhos/ FIOCRUZ, Rio de Janeiro, Brazil) that uses the recombinant protein K39 (rK39) as an antigen, a cloned 39 amino acid sequence of the specific *L. infantum* kinase region, was performed according to the manufacturer's guidance.

#### **2.4 DNA extraction, conventional PCR, and qPCR**

DNA extraction from blood samples was performed by the phenol-chloroform method. The polymerase chain reaction assay was performed using the primers RV1 (sense) 5′-CTT TTC TGG TCC CGC GGG TAG G-3′ and RV2 (antisense) 5′-CCA CCT GGC TAT TTT ACA CCA-3′ [24], which amplifies the DNA fragment of a 145 bp region of conserved kDNA present in *L. infantum*. Amplification used 200 mM dNTP, 1 pM from each primer, a buffer solution (10 mM Tris–HCl and 50 mM KCl, pH 8.3), 2 mM MgCl2, 1.5 U Taq DNA polymerase, and 2 μl of the DNA sample in the final volume of 25 μl. Assays were performed for one cycle at 94°C for 4 minutes, followed by 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and final extension of one cycle at 72°C for 10 minutes. The amplification product was fractionated by 2.0% agarose gel electrophoresis, stained with red gel spot, and visualized on a transilluminator (UV, 300 nm).

Quantitative PCR (qPCR) was performed in triplicate using the StepOne™ Real-Time PCR System Sequence Detection System (Applied Biosystems) targeting RV1–5′-CTT TTC TGG TCC GGG TAG G-3′ primers and RV2–5′-CCA CCT GGC TAT TTT ACA CCA-3′ amplifying a 145 bp sequence of *L. infantum-*specific kDNA [24]. Reactions were prepared in a 25 μl final volume containing SYBR Green Master Mix, 0.3 μM of each primer, and 2 μl of target DNA. Amplification conditions included an initial incubation step at 94°C for 10 minutes, followed by 40 cycles of amplification, 94°C for 15 seconds, and 60°C for 60 seconds. The standard curve was established for each assay using known amounts of TOPO PCR 2.1 plasmid (Invitrogen Corp.) containing *L. infantum* kDNA gene. Serial (10×) dilutions of the recombinant plasmid containing 2.9×104 –2.9×108 copies of the plasmid were performed and used on the standard curve.

#### **2.5 Cytokine quantification by flow cytometry**

Serum cytokine concentration (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was assessed using the Cytometric Bead Array (CBA) Kit (BD Bioscience, USA) and evaluated by a flow cytometer (FACSCalibur®, BD Bioscience, USA). The reading was done using the CellQuest. Data were analyzed in FCAP array software version 5.0.

#### **2.6 Determination of serum immunoglobulins**

Immunoglobulin concentrations (IgM and IgG) in the sera were determined by turbidimetric method. For 1:11 (v/v) IgM and 1:15 (v/v) IgG, antibody concentrations were determined using IgM (Bioclin®, Brazil, Ref K063) and IgG (Bioclin®, Brazil, Ref K062) antiserum diluted with 1:12 (v/v). The calibration curve obtained from the Multical calibrator (Bioclin®, Brazil, Ref K064) was used to determine the standard curve for each immunoglobulin. Positive and negative serum samples, standards, and controls were placed in 500 μl buffer solution (0.15 mol/L sodium

chloride, Tris 50 mmol/L, 6.0000 PEG 50 g/L, and sodium azide 15.38 nmol/L). The suspensions were mixed and incubated at 37°C for 10 minutes. Reactions were read on a spectrophotometer at 340 nm.

#### **2.7 Statistical analysis**

For the analysis of the concentration of cytokines and immunoglobulins (IgG and IgM), the Student t-test independent samples were used. For the quantification of parasitic index of the bone marrow and cytokines when compared by clinical stage, Kruskal-Wallis analysis of variance was used. Parasite load correlation analysis of IgG in the presence of cytokines was also performed by calculating the Spearman correlation coefficient. Data were expressed as mean ± standard error. Values less than 0.05 (p < 0.05) were considered significant.

## **3. Results**

Most of the 21 dogs in the control group were mongrel dog (15/71%), Labrador retriever (1/5%), dachshund (1/5%), pinscher (3/14%), and rottweiler (1/5%). Age ranged from 14 months to 8 years (average 3.4 years). Thirteen dogs were female (13/62%) and eight dogs were male (8/38%). Most of the 21 dogs with leishmaniasis were dogs from mongrel dog (12/57%), Labrador retriever (1/05%), American pit bull (1/05%), poodle (1/05%), and shih tzu (6/28%). Age ranged from 12 months to 11 years (mean 4.3 years). Six dogs were female (6/29%) and 15 dogs were male (15/72%).

At the time of clinical evaluation, all dogs diagnosed with VL had several clinicopathological findings typical of the disease. Clinical symptoms in seropositive animals (CVL) included lymphadenopathy (17/13%), skin ulcers (12/10%), onychogryphosis (11/09%), ear ulceration (11/09%), scaling (10/08%), weight loss (9/07%), dermatopathy (8/06%), ophthalmopathy (8/06%), muscle atrophy (4/03%), splenomegaly (7/06%), alopecia (6/05%), lethargy (5/04%), periocular alopecia (4/03%), skin nodules (3/02%), hepatomegaly (3/02%), cachexia (3/02%), and hyperkeratosis (2/01%).

Dogs were classified into three clinical stages: stage I, mild disease (n = 5/24%); stage II, moderate disease (n = 9/43%); and stage III, severe disease (n = 7/33%). Stage II dogs were not subclassified.

*Leishmania infantum* DNA was detected in all dogs of the group with CVL up to a concentration of 1 fg/μl. Real-time PCR of bone marrow samples was positive in all dogs in the CVL group (100%). There was no statistical difference in the distribution between clinical stages and parasitic index, as shown in **Table 1**.

The mean and standard error of concentrations (pg/ml) of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17 cytokines based on clinical staging in CVL-infected dogs are shown in **Table 1**. It was observed that IL-6 and TNF-α concentrations increased in serum of infected dogs with significant statistical difference between the clinical stages of CVL, although most infected dogs had moderate and severe clinical manifestations of the disease.

Among dogs with CVL and uninfected dogs, an increase of IL-4 and TNF-α concentrations in serum from dogs infected with CVL was observed. Similar serum concentrations of IL-2, IL-10, IL-17, and IFN-γ were observed between the groups studied (**Table 2**).

When comparing immunoglobulin means, IgG levels were elevated in the CVL group when compared to IgM levels. A significant difference (p = <0.0001)

**107**

**Table 2.**

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis*

was observed. Similarly, IgG concentration between the control and CVL groups was evaluated. IgG levels were found to be higher in serum from dogs with CVL (2300.75 ± 678.463) when compared to control group IgG concentrations (636.94 ± 312.8 mg/dl), showing a significant difference between groups (p = <0.0001). Regarding the comparison of IgM concentration (mg/dl) in the CVL group (279.74 ± 37.755) compared to the control group (241.12 ± 59.835), there was

Correlations of IL-6 and TNF-α concentrations were analyzed according to clinical staging with parasitic index according to stage I, IL-6 (rs = 0.400, p = 0.5046) and TNF-α (rs = 0.700, p = 0.1881); stage II, IL-6 (rs = 0.7000, p = 0.1881) and TNF-α (rs = −0.1590, p = 0.6828); and stage III, IL-6 (rs = −0.3571, p = 0.4316) and TNF-α (rs = −0.4643, p = 0.2939). There was no correlation between the other

The correlation between the parasitic index of dogs with CVL in the presence of cytokine IL-4 and TNF-α in the blood of dogs infected with CVL presented the IL-4 (rs = 0.0240, p = 0.9176) and TNF-α (rs = 0.0825, p = 0.7221). No additional significant correlations were found. Antibody levels were positively correlated with

**Cytokines/parasitemia I II III p-Value** IL-2 6.62 ± 1.18 12.01 ± 7.99 15.09 ± 6.34 0.152 IL-4 10.50 ± 2.05 11.38 ± 3.81 9.90 ± 2.73 0.9044 IL-6 2.14 ± 0.57 2.72 ± 0.66 3.12 ± 0.50 0.0350 IL-10 2.47 ± 0.97 2.85 ± 0.96 2.39 ± 0.84 0.8973 IL-17 2.22 ± 0.22 12.38 ± 9.63 13.27 ± 7.51 0.4345 TNF-α 4.52 ± 2.12 4.65 ± 2.31 6.14 ± 1.43 0.0462 IFN 3.07 ± 0.99 28.19 ± 23.21 2.58 ± 0.28 0.4648

**Cytokines Control CVL p-Value** IL-2 9.18 ± 6.14 11.75 ± 6.89 0.3199 IL-4 7.43 ± 2.50 12.56 ± 5.37 0.0469 IL-6 2.87 ± 0.95 2.71 ± 0.67 0.3326 IL-10 2.98 ± 1.39 2.62 ± 0.87 0.2807 IL-17 11.12 ± 12.12 11.63 ± 9.66 0.4570 TNF-α 2.80 ± 0.52 5.12 ± 2.33 0.0009 IFN 13.26 ± 16.88 16.15 ± 19.01 0.3589

)/ml 4.96 ± 1.00 4.63 ± 1.37 4.55 ± 1.49 0.9467

*)/ml in dogs with visceral leishmaniasis in different* 

*DOI: http://dx.doi.org/10.5772/intechopen.90573*

IL-4 expression (rs = 0.5997, p = 0.0040) (**Table 4**).

no difference (**Table 3**).

parameters evaluated.

Parasite copy number (×107

**Table 1.**

*clinical staging.*

*The results were expressed in mean and standard error.*

*Cytokine concentrations and parasite copy number (×107*

*The results were expressed in mean and standard error.*

*Cytokine concentrations in dogs noninfected and dogs with canine visceral leishmaniasis.*

*Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.90573*

was observed. Similarly, IgG concentration between the control and CVL groups was evaluated. IgG levels were found to be higher in serum from dogs with CVL (2300.75 ± 678.463) when compared to control group IgG concentrations (636.94 ± 312.8 mg/dl), showing a significant difference between groups (p = <0.0001). Regarding the comparison of IgM concentration (mg/dl) in the CVL group (279.74 ± 37.755) compared to the control group (241.12 ± 59.835), there was no difference (**Table 3**).

Correlations of IL-6 and TNF-α concentrations were analyzed according to clinical staging with parasitic index according to stage I, IL-6 (rs = 0.400, p = 0.5046) and TNF-α (rs = 0.700, p = 0.1881); stage II, IL-6 (rs = 0.7000, p = 0.1881) and TNF-α (rs = −0.1590, p = 0.6828); and stage III, IL-6 (rs = −0.3571, p = 0.4316) and TNF-α (rs = −0.4643, p = 0.2939). There was no correlation between the other parameters evaluated.

The correlation between the parasitic index of dogs with CVL in the presence of cytokine IL-4 and TNF-α in the blood of dogs infected with CVL presented the IL-4 (rs = 0.0240, p = 0.9176) and TNF-α (rs = 0.0825, p = 0.7221). No additional significant correlations were found. Antibody levels were positively correlated with IL-4 expression (rs = 0.5997, p = 0.0040) (**Table 4**).


#### **Table 1.**

*Parasitology and Microbiology Research*

on a spectrophotometer at 340 nm.

**2.7 Statistical analysis**

**3. Results**

(15/72%).

and hyperkeratosis (2/01%).

Stage II dogs were not subclassified.

clinical manifestations of the disease.

chloride, Tris 50 mmol/L, 6.0000 PEG 50 g/L, and sodium azide 15.38 nmol/L). The suspensions were mixed and incubated at 37°C for 10 minutes. Reactions were read

For the analysis of the concentration of cytokines and immunoglobulins (IgG and IgM), the Student t-test independent samples were used. For the quantification of parasitic index of the bone marrow and cytokines when compared by clinical stage, Kruskal-Wallis analysis of variance was used. Parasite load correlation analysis of IgG in the presence of cytokines was also performed by calculating the Spearman correlation coefficient. Data were expressed as mean ± standard error.

Most of the 21 dogs in the control group were mongrel dog (15/71%), Labrador retriever (1/5%), dachshund (1/5%), pinscher (3/14%), and rottweiler (1/5%). Age ranged from 14 months to 8 years (average 3.4 years). Thirteen dogs were female (13/62%) and eight dogs were male (8/38%). Most of the 21 dogs with leishmaniasis were dogs from mongrel dog (12/57%), Labrador retriever (1/05%), American pit bull (1/05%), poodle (1/05%), and shih tzu (6/28%). Age ranged from 12 months to 11 years (mean 4.3 years). Six dogs were female (6/29%) and 15 dogs were male

At the time of clinical evaluation, all dogs diagnosed with VL had several clinicopathological findings typical of the disease. Clinical symptoms in seropositive animals (CVL) included lymphadenopathy (17/13%), skin ulcers (12/10%), onychogryphosis (11/09%), ear ulceration (11/09%), scaling (10/08%), weight loss (9/07%), dermatopathy (8/06%), ophthalmopathy (8/06%), muscle atrophy (4/03%), splenomegaly (7/06%), alopecia (6/05%), lethargy (5/04%), periocular alopecia (4/03%), skin nodules (3/02%), hepatomegaly (3/02%), cachexia (3/02%),

Dogs were classified into three clinical stages: stage I, mild disease (n = 5/24%); stage II, moderate disease (n = 9/43%); and stage III, severe disease (n = 7/33%).

*Leishmania infantum* DNA was detected in all dogs of the group with CVL up to a concentration of 1 fg/μl. Real-time PCR of bone marrow samples was positive in all dogs in the CVL group (100%). There was no statistical difference in the distribu-

The mean and standard error of concentrations (pg/ml) of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17 cytokines based on clinical staging in CVL-infected dogs are shown in **Table 1**. It was observed that IL-6 and TNF-α concentrations increased in serum of infected dogs with significant statistical difference between the clinical stages of CVL, although most infected dogs had moderate and severe

Among dogs with CVL and uninfected dogs, an increase of IL-4 and TNF-α concentrations in serum from dogs infected with CVL was observed. Similar serum concentrations of IL-2, IL-10, IL-17, and IFN-γ were observed between the groups

When comparing immunoglobulin means, IgG levels were elevated in the CVL group when compared to IgM levels. A significant difference (p = <0.0001)

tion between clinical stages and parasitic index, as shown in **Table 1**.

Values less than 0.05 (p < 0.05) were considered significant.

**106**

studied (**Table 2**).

*Cytokine concentrations and parasite copy number (×107 )/ml in dogs with visceral leishmaniasis in different clinical staging.*


#### **Table 2.**

*Cytokine concentrations in dogs noninfected and dogs with canine visceral leishmaniasis.*


#### **Table 3.**

*Immunoglobulin concentrations (IgG and IgM) in serum from dogs with canine visceral leishmaniasis.*


*rs, correlation coefficient of Spearman.*

#### **Table 4.**

*Correlation between IgG concentrations with IL-4 and TNF-α and parasitic index of dogs infected with CVL.*


#### **Table 5.**

*Correlation of IL-6 and TNF-α cytokine levels of dogs with canine visceral leishmaniasis by clinical staging of serum from dogs of the CVL group.*

In this study, as shown in **Table 5**, the correlation of the evolution of clinical signs between the stages presented below was analyzed. There was a significant positive correlation of IL-6 cytokine levels between stage I and stage III.
