**4. 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

**109**

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

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

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 (×1010)/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

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

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%

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

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

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

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

between healthy and sick dogs [27].

high parasitic index is detected [21].

and specificity of 96.4% [30].

dogs [32] as used in this study.

literary quote converges with the findings of this study.

only for cytokines IL-6 (p = 0.0350) and TNF-α (p = 0.0462).

change from an acute inflammatory state to a chronic state [37].

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

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 (×1010)/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].

*Parasitology and Microbiology Research*

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

*rs, correlation coefficient of Spearman.*

*rs, correlation coefficient of Spearman.*

*serum from dogs of the CVL group.*

**Table 3.**

**Table 4.**

**Table 5.**

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

*Correlation of IL-6 and TNF-α cytokine levels of dogs with canine visceral leishmaniasis by clinical staging of* 

**Group Control CVL p***-***Value** IgG 636.94 ± 255.52 2288.04 ± 610.08 <0.0001 IgM 241.12 ± 51.81 282.42 ± 33.99 0.0773

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

**Parameters rs p-Value** IL-4 0.5997 0.0040 TNF-α 0.4164 0.0603 Parasitic index −0.2243 0.3282

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

**Stage I and II I and III II and III**

IL-6 0.6031 0.0855 0.8469 0.0162 0.5630 0.1144 TNF-α 0.0350 0.9288 0.3784 0.4026 0.0168 0.9658

**rs p rs p rs p**

**IgG**

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

positive correlation of IL-6 cytokine levels between stage I and stage III.

**108**

**4. Discussion**

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].

#### **5. 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

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

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

This research received grants from the Mato Grosso Research Support Foundation (FAPEMAT No. 299032/2010) and from the National Council for Scientific and Technological Development (CNPq No. 447218/2014–0 and No.

The authors declare that there is no conflict of interest and nonfinancial

, Valéria Régia Franco Sousa1

1 Faculty of Veterinary Medicine, Federal University of Mato Grosso, Cuiabá, Mato

2 Institute of Biological and Health Sciences, Federal University of Mato Grosso,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

,

, Adenilda Cristina Honorio-França2

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

**Acknowledgements**

305725/2018–1), in Brazil.

**Conflict of interest**

competitors.

**Author details**

Grosso, Brazil

José Nivaldo da Silva1

and Eduardo Luzía França2

Arleana do Bom Parto Ferreira de Almeida1

Barra do Garças, Mato Grosso, Brazil

provided the original work is properly cited.

\*

\*Address all correspondence to: elfranca@ufmt.br

marker to distinguish between different clinical stages.

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

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.
