Relationship of Parasitic Index and Cytokine Profile in Canine Visceral Leishmaniasis

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. (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%).

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].

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.

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.

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.

Immunochromatographic rapid test: RT DPP® kit
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.

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 2.9×10 4 -2.9×10 8 copies of the plasmid were performed and used on the standard curve.

Determination of serum immunoglobulins
Immunoglobulin concentrations (IgM and IgG) in the sera were determined by turbidimetric method. ) 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.

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.
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)  Table 3).
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) ( The results were expressed in mean and standard error.  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.

Discussion
In this study the most dogs in the control group and CVL were mixed breed. The clinical symptoms of seropositive dogs (CVL) included lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, scaling, weight loss, and others. Dogs were classified into three clinical stages: stage I, mild disease; stage II, moderate disease; and stage III, severe disease. There was no statistical difference in the distribution between clinical stages and parasitic index. IL-6 and TNF-α concentrations increased in serum from infected dogs with a statistically significant difference between the clinical stages of CVL. Between the dogs with CVL and the control group, there was a statistical difference in the serum concentrations of cytokines IL-4 and TNF-α. IgG levels were elevated in the CVL group when compared to IgM levels. Antibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). There was a significant positive correlation of IL-6 cytokine levels between stage I and stage III.
The clinical signs of CVL are important for the diagnosis. In the present study, the most prevalent clinical signs were lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, and scaling. However, prevalence is highly variable across    studies, but generally these clinical signs are the most commonly reported in the literature. These results corroborate the findings of several authors [25,26]. Regarding gender, there was a greater predominance of males in infected dogs and females in dogs in the control group. Regarding age, it did not present large variations. This fact seems to be associated with the higher risk of male exposure. However, the study shows no statistically significant differences for age and gender between healthy and sick dogs [27].
Bone marrow samples were taken from 21 dogs serologically positive for L. infantum. According to the clinical signs, dogs were classified as stages I, II, and III. Real-time PCR detected no parasite copies (×10 10 )/μl L. infantum DNA in all animals of the CVL group, distributed as follows: stage I mean (4.964), stage II average (4.63), and stage III (4.55). No statistically significant difference was found in the average amount of DNA copy number between the different clinical stages (p = 0.9467). In bone marrow samples from dogs that are cytologically positive, a high parasitic index is detected [21].
Previous studies report that quantitative PCR on bone marrow samples from positive dogs in conventional tests contained a higher number of Leishmania kDNA copies than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load [28]. This literary quote converges with the findings of this study.
PCR can be used for detection of Leishmania in naturally infected dog samples, and PCR-RFLP (restriction fragment length polymorphism) is sensitive for identification of Leishmania species [28]. In addition, qPCR is effective in quantifying Leishmania DNA loading in clinical samples [29]. The blood sample from dogs infected by L. infantum was found by real-time PCR to have a sensitivity of 100% and specificity of 96.4% [30].
Most cytokines remain partially conserved between species; in this sense, the amino acid sequence of humans and canine cytokines shows 49-96% homology, suggesting a high probability of cross-reactivity between monoclonal antibodies; thus antibodies against human cytokines may be recommended as immunological biomarkers under pathological conditions by flow cytometry in human [31] and dogs [32] as used in this study.
In the present work, the serum concentration of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was compared between the control groups and the group with CVL. In addition, cytokine levels were compared within the CVL group with clinical staging I, II, and III. When comparing the groups, IL-4 and TNF-α were higher in infected dogs than in the control group, showing significant difference between IL-4 (p = 0.0469) and TNF-α (p = 0.0009) groups. In the group with CVL there were differences between stages I and III with significant differences only for cytokines IL-6 (p = 0.0350) and TNF-α (p = 0.0462).
Elevated levels of IL-6 were found in serum from dogs with active leishmaniasis compared to healthy dogs [33]. These results corroborate the findings of this study. However, other authors reported that IL-6 production did not vary significantly between the groups studied [34]. On the other hand were described in the literature that elevated levels of IL-6 in dogs without clinical signs or symptoms in CVL dogs [35], and also highlights that, among other factors, it may indicate a balance between the parasite elimination effort and the active disease. Increased IL-6 levels suggest a restricted ability to control infection [36]. Even in the absence of clinical signs or symptoms, the animals showed granulomas on histopathological evaluation, suggesting chronicity and therefore a longtime course of infection [35]. Innate immune effector cells primarily neutrophils, monocytes, and macrophages produce and respond to IL-6, which may result in amplification of inflammation and a change from an acute inflammatory state to a chronic state [37]. IL-6 expression increases in dogs with active visceral leishmaniasis and may be a useful marker for active disease [33,35]. Increased IL-6 production is not directly related to anti-Leishmania antibody titers, suggesting that other cytokines may be involved with hypergammaglobulinemia [33].
As shown in this work, it was observed that there was correlation of IL-6 expression between stages I and III of bone marrow aspirate of dogs infected with CVL. IL-6 production in dogs with active leishmaniasis appears to be associated with severe disease [33]. This statement converges with the findings in this study, as the dogs used in the control group were mostly stage II and III. IL-6 is essential for terminal B-cell differentiation and immunoglobulin production [38].
TNF-α concentration was higher in infected dogs than in the control group, as detected by de Lima et al. [33]. CVL susceptibility is closely associated with downregulation of key cytokines such as IFN-γ, TNF-α, and IL-17A, thus impairing iNOS activation and NO production and favoring parasite replication and disease development [39].
The increased activity of TNF-α in the liver of infected dogs compared to healthy canines has been reported [37,40]. Higher TNF-α levels in infected dogs indicate that the presence of L. infantum induces an immune response with relevant TNF-α expression when the protozoan is present [40].
Studies suggest that decreased survival of L. infantum in canine macrophages is associated with increased TNF-α and IFN-γ production and decreased IL-10 production [41].
In dogs naturally infected with L. infantum, increased hepatic TNF-α may be associated with increased parasite load on this organ [42]. The cytokines IL-2, IL-4, IL-10, IFN-γ, TNF-α, and IL-12 may be used as markers in epidemiological studies conducted in endemic areas to distinguish between different clinical forms of VL [15]. However, Lima et al. [33] indicate that TNF-α is not considered a good marker of active disease in dogs with VL.
A study has reported a significant relationship between bone marrow IL-4 detection in naturally infected dogs with and without clinical signs and disease severity, suggesting that IL-4 production is associated with pathology [43]. Increased expression of IL-4 cytokine is associated with both severe clinical signs and a high parasitic index on skin lesions [44]. In bone marrow aspirates, IL-4 was elevated in naturally infected dogs with more severe symptoms [43].
The study points to evidence that IL-4 cytokine polymorphism may contribute to innate immunity to L. infantum infection [45].
Antibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). IgG is also linked to chronic infection in patients with VL, where high levels of IgG are predictive of the disease. This finding is in line with the study by Lima et al. [33] suggesting that other cytokines, such as IL-10 or IL-4, may be associated with hypergammaglobulinemia observed in dogs with CVL. Previous studies have detected increased serum IgG levels in symptomatic dogs compared with healthy dogs and are related to pathophysiological disorders and active disease [33].
Response to natural infection of L. infantum is linked to the presence of IgG [43] and Leishmania-specific IgM antibodies that can be detected in infected dogs [46]. Some studies have reported that increased total protein is frequent in dogs infected with visceral leishmaniasis due to increased antibody production [47,48].

Conclusion
These results may contribute to a better understanding of the immune response in dogs infected with L. infantum. Antibody levels were positively correlated with Xavier AAP, Gontijo CMF. Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR. Acta Tropica. 2009;111:289-294