**4. Clinical signs and symptoms**

After evaluating expression of cytokines in spleen cells from dogs naturally infected with *L. infantum*, Lage et al. suggested that CVL is characterized by a mixed response: with production of cytokines types I and II; involvement of IFN-y and IL-10 and a positive correlation between IL-10 levels and progression of parasitic load or clinical manifestations of the disease; and correlation between IFN-γ and increased parasitic intensity of the spleen [84]. In whole blood, the increase of IL-10 has been associated with detection of parasite DNA [97]. Do Nascimento et al., in accordance, state that in CVL there is increase in expression of pro-inflammatory cytokines IFN-γ and TNF-α in dogs with low splenic parasitism, while dogs with higher

Souza [99] states that asymptomatic dogs have low dermal parasitism and exhibit a mixed pattern of immune response, with simultaneous increase of type I (IFN-γ and TNF-α) and type II (IL-5 and IL-13) cytokines, but predominance of type I response. According to the author, increased and simultaneous expression of IFN-γ and TNF-α in the skin of infected dogs enables the speculation that these mediators are closely involved with protection mechanisms during CVL, since these cytokines increased in the skin of animals with the asymptomatic clinical form. Increased expression of IL-5 and IL-13 in the skin of healthy dogs and negative correlation of the latter with clinical disease progression were also observed. Furthermore, high simultaneous expression of IFN-γ and IL-13 was found in asymptomatic dogs, indicating the

Regarding cytokine profile in the bone marrow, Quinnell et al. [94] reported that expression of mRNA for IL-10, IL-4, and IL-18 was not elevated in infected dogs. However, some infected dogs had detectable expression of mRNA for IL-4 significantly correlated with more severe clinical signs. Moreover, expression of mRNA for IL-13 was not detected either in control or in infected dogs, and unlike in human infection, immunosuppressive activity of IL-10 was not

Dogs infected with *L. infantum* exhibit significant decrease in expression of mRNAs for IL-10, IL-17, TNF-α, IFN-γ, and iNOS in liver tissue. Deficiency in IL-17 mRNA expression was evident in the symptomatic dogs compared to the asymptomatic. Reduction in cytokine expression results in decreased iNOS expression and therefore higher parasite load. The increase in IL-17 expression in the liver of asymptomatic dogs and its correlation with elevated expression of iNOS indicates a protective role of that cytokine in canine infection by *L. infantum* [67]. However, Michelin et al. [100] reported increased TNF-α, IL-4, and IL-10 levels in the liver of infected dogs compared to healthy dogs. In addition, the association between TNF-α levels and an increase in parasitic load in the spleen suggests its importance in the evolution of the

Another subject lacking clarification is the participation of chemokines and their receptors in resistance or susceptibility to LVC. Knowledge surrounding the role of these modulators in response to *L. infantum* infection is critical, considering that the interaction between cytokines and chemokines may regulate the immune response against the parasite, activating and

parasitic load show an increase in IL-10 [98].

30 Canine Medicine - Recent Topics and Advanced Research

role of IL-13 in establishing milder clinical forms [99].

observed in CVL [94].

infection process [100].

recruiting immune cells to areas of infection.

Canine visceral leishmaniasis presents a clinical picture ranging from asymptomatic to classical symptomatic cases (**Figure 4**). Infected animals without clinical signs comprise an

**Figure 4.** Classical symptomatic case with emaciation, thickened skin, cutaneous lesions, exfoliative dermatitis, fur loss, and cutaneous ulcers.

alarming 40–80% of all cases, for asymptomatic dogs are a major source of infection for sand flies, and owners naturally resist to elimination of their animals [102].

At first, kala-azar signals can be rather discrete and easily confused with other diseases. Animals may have discrete lesions on the edge of the ears and slight changes in blood profile (mild anemia and/or thrombocytopenia).

Clinical leishmaniasis may appear quickly after infection or within two years. Classic canine kala-azar is characterized by thickened skin, cutaneous lesions, intermittent fever, appearance shift and fur loss, periorbital alopecia, hepatosplenomegaly, akinesia, diarrhea, onychogryphosis (nail growth; **Figure 5**), and nosebleeding. Partial paralysis of hindquarters is often seen in the final stage of the disease.

**Figure 5.** Onychogryphosis.

The most common skin lesions in dogs with kala-azar are exfoliative dermatitis (generalized, regional, or localized); ulcerative dermatitis, onychogryphosis, and papular dermatitis.

Cutaneous ulcers are usually located on the ear margins and have been attributed to local trauma and/or vasculitis, pressure points (**Figure 6**), limbs, and mucocutaneous junctions. Focal or multifocal nodular forms have a high amastigote load and may indicate either inefficient or strong cellular immunity by the host [101, 103–105].

Ocular disease occurs in CVL, with anterior uveitis being the most common ocular manifestation, characterized by conjunctivitis, blepharitis, periocular alopecia, exophthalmia, keratitis, keratoconjunctivitis sicca, anterior uveitis, glaucoma, and retinal detachment [106].

The nosebleeding (epistaxis) occurs due to thrombocytopenia and is often confused with ehrlichiosis, a bacterial disease transmitted by ticks, which in many cases might associate with leishmaniasis. In endemic areas, it is advisable that any diagnosis of ehrlichia or anaplasma in dogs must be accompanied by differential diagnosis of kala-azar.

In general, dogs in endemic areas are poly-infected and malnourished, particularly stray dogs or those who frequently wander on the streets, leading to a plurality of overlapping clinical pictures. Among other conditions, furfuraceous flaking due to scabies, weight loss as consequence of other infections or lymphomas, and autoimmune diseases, such as systemic lupus erythematosus, often confuse diagnosis since clinical signs are usually not pathognomonic. Therefore, differential diagnosis must be a concern for the small animal clinician [107, 108].

**Figure 6.** Cutaneous ulcer located on a pressure point.
