**3. Immunology**

The components of the immune system act in a complex and coordinated manner to prevent entry and survival of foreign agents in the body. The first line of defense is the innate immunity, which responds immediately and unspecifically to a range of pathogens, and further presents them to the constituents of adaptive immunity when needed. Adaptive immunity will then generate a specific response and develop memory cells against such antigen. Performance of these defense mechanisms can control infection and ensure the least possible damage to host tissues.

In visceral leishmaniasis, the result of the relationship between parasite and host is determined by complex factors involving saliva components of the vector insect, agent-secreted surface proteins, and different responses produced by the host [67]. Leishmaniasis can be considered an immune-mediated disease, considering the parasite's ability to alter the immune system [68]. It ultimately promotes inhibition of immune response by either stimulating the development of regulatory T cells [69] or exerting some degree of control over the complement system, exploring its opsonic properties to facilitate adherence with phagocytic cells and preventing their lytic effects through the action of gp63 glycoprotein expressed on the parasite surface [70]. Thus, infection outcome depends on the parasite's capacity of developing evasion mechanisms to escape from host responses and remain unharmed in the cytoplasm of phagocytic cells.

It has been documented that resistance to infection by *L. infantum* in dogs is characterized by the absence of clinical signs, low levels of anti-*Leishmania* antibodies, in vitro lymphocyte proliferative response, and delayed hypersensitivity response to skin antigens. Progression of the infection, however, relates to exaggerated humoral response and cellular immune depression, consequently bringing up an onset of clinical signs [71–74]. Asymptomatic animals also exhibit lower parasitism, whereas the symptomatic generally carry high parasite load in different tissues such as the skin, bone marrow, spleen, liver, and lymph node [73–76].

#### **3.1. Innate response**

The best example of the phenomenon of urbanization of zoonotic visceral leishmaniasis is happening in Brazil [54, 63]. VL has invaded urban centers and large capitals with no previous record of autochthonous cases [12]. Epidemiological data show the suburbanization and urbanization of visceral leishmaniasis, highlighting the outbreaks in Rio de Janeiro (RJ), Belo Horizonte (MG), Aracatuba (SP), Santarém (PA), Corumbá (MS), Teresina (PI), Natal (RN), São Luís (MA), Fortaleza (CE), Camaçari (BA), and more recently, occurrence of epidemics in the

The prevalence of human VL caused by *L. infantum* has decreased where living standards improved [2], showing that one of the most important interventions may be socioeconomic

There is no consensus on the risk factors associated with CVL, as results differ between the

In Croatia, risk factors were sex (male), age (the two most prevalent groups comprise dogs between 3 and 4 years old and between 6 and 7 years old), and location (dogs in some cities are more likely to acquire the disease) [30]. In Spain, seroprevalence was also found to have bimodal age distribution, but the age groups were between 1 and 2 years and between 7 and 8 years; infection is also related to outdoor rearing [64]. In Portugal, risk factors are outdoor rearing, age (over two years), short fur, pure breeds, and location (dogs in the hinterlands are

In Brazil, Belo et al. [62] conducted a systematic review of the literature on risk factors associated with CVL, and the variables that showed significant association with infection were short hair, pure breed, rearing restricted to house surroundings, and the presence of green areas adjacent to the house. The occurrence of CVL was also associated with the presence of poultry in domestic environment, free-living dogs, sex (male), and age greater than 1 or 2 years,

Another study in Brazil defined risk factors as outdoor rearing, contact with poultry, dogs living in rural areas, the presence of organic matter, the absence of environmental management, and proximity to forests [65]. As for dogs in the countryside, in an endemic area of Northeastern Brazil, the only identified risk factor found was sex, as male dogs were twice as likely to develop

The components of the immune system act in a complex and coordinated manner to prevent entry and survival of foreign agents in the body. The first line of defense is the innate immunity, which responds immediately and unspecifically to a range of pathogens, and further presents them to the constituents of adaptive immunity when needed. Adaptive immunity will then generate a specific response and develop memory cells against such antigen. Performance of

municipalities of Três Lagoas (MS), Campo Grande (MS), and Palmas (TO) [11].

development and improved nutrition of children [8], but CVL cases have increased.

**2.5. Risk factors**

the disease [66].

**3. Immunology**

more likely to be affected) [29].

studied Brazilian regions and between countries.

26 Canine Medicine - Recent Topics and Advanced Research

although these associations were not statistically significant [62].

Through antigen presentation, the cells of innate immunity stimulate the acquired response. Antigen-presenting cells have receptors that recognize pathogen-associated molecular patterns (PAMPs) expressed by the parasite. Among these, the Toll-like receptor (TLR) is one of the most studied. Stimulation of these receptors culminates with the activation of signaling pathways in infected cells, which results in induction of antimicrobial genes and inflammatory cytokines (IL-12, TNF) while increasing the ability of cells to present antigen. Thus, pathogen recognition by TLR receptors helps conducting adaptive immune response against the presented antigen [77].

Expression of TLR genes in dogs infected with *L. infantum* varies according to the different tissues and stages of infection. As the disease progresses, there is a significant decrease in transcription for TLR3, TLR4, TLR9, IL-17, IL-22, and FoxP3 in lymph nodes. In spleen samples, decreased transcription for TLR4 and IL-22 has been observed when infected groups were compared with controls. In the skin, upregulation was observed only for TLR9 and FoxP3 in early stages of infection, as well as downregulation for TLR3 and TLR9 in later stages. Decrease in transcription of TLRs, Th17, and FoxP3 cytokines is suggestive of silent establishment of infection [78]. In peripheral blood samples from infected dogs, the highest expression of TLR2 and its receptor CD11b (CR3) by monocytes correlates with reduced parasitic load and higher resistance to leishmaniasis [79].

Studies have shown that both inlet and survival of *Leishmania* spp. within macrophages can occur from prior infection of neutrophils, which are recruited as a normal response to insect bites. The agent reaches the interior of macrophages when they phagocyte apoptotic bodies of previously infected neutrophils, where it can survive and multiply. This parasite escape mechanism is called "Trojan Horse" [80].

The main effector mechanism involved in protective immune response against *Leishmania* spp*.* is the activation of macrophages by IFN-γ and tumor necrosis factor alpha (TNF-α) stimulation, with consequent stimulation of nitric oxide synthesis, which is required for effective destruction of the pathogen and for controlling the spread of infection in dogs [81–83].

## **3.2. Acquired response**
