**5.1. Chemotherapy**

Due to the possibility of parasitic resistance to first-choice drugs, chemotherapeutic treatment options are limited. In addition, due to the high cost and long-term use, chemotherapy becomes an undesirable option for owners.

In veterinary medicine, the first choice as chemotherapy for treatment of CVL is allopurinol, a leishmaniostatic drug that acts by inhibiting leishmania growth through DNA modification. It has low cost, but parasite resistance to it remains unknown. Allopurinol is however the only drug recommended by the World Health Organization, especially since it is little used for treatment of human leishmaniasis [113].

Antimonials are leishmanicidal drugs that hinder promastigote metabolism by inhibiting glycolytic activity. These are drugs of choice for human treatment of VL but are chemically similar to those that have been used in therapeutic protocols in dogs. Their toxicity and efficacy are related to the antimony content [113].

When assessing the therapeutic efficacy of Glucantime alone or associated with an antigenic extract of *L. braziliensis* in experimentally infected asymptomatic dogs, reduction in parasite burden and antibody levels decreased, but actual cure was not achieved. Yet, upon using meglumine antimoniate and allopurinol—alone and in combination—in dogs naturally infected by *L. infantum*, symptomatic animals showed remission of clinical signs about 60 days after the treatment, accompanied by restoring of normal urinary and hematologic profiles. Despite clinical improvement of treated animals, however, the combination was not able to eliminate the parasite in all dogs. Thus, this protocol is not recommended, since treated dogs would go on as a source of infection for humans and other dogs, similar to results obtained using antimoniate, which promoted hematologic normalization and bone marrow recovery, but not parasitological cure, and to findings from another study wherein dogs showed increased parasitic load in lymph nodes [114–117].

In accordance with Ikeda-Garcia et al. [116], Manna et al. [118] monitored leishmania DNA load in infected dogs through real-time PCR, using a similar treatment protocol. Therapy resulted in clinical improvement accompanied by reduction in parasite burden, but even after a long period of treatment with allopurinol alone, parasites remained in tissues [116, 118].

Miltefosine is another drug used in human treatment that has been evaluated for canine treatment in recent years. It is a phospholipid antibiotic of broad spectrum with leishmanicide effect that improves the activation of both macrophages and T cells [119]. After evaluating efficacy of three treatment protocols for dogs naturally infected with *L. infantum*, significant clinical improvement was observed and later progressed to full recovery and humoral stimulation, but did not assure cure, on the contrary, progressively increased parasite load. Hence, miltefosine alone is not recommended for treatment. The study also investigated the profile of major cytokines involved in CVL healing process, and findings showed an increase of IFN-γ within 90 days after treatment, when the parasite load in bone marrow aspirates also decreased. There was a decrease of IL-4, indicating possible resolution of the infection and efficacy of the treatment, since this cytokine is a good marker for the occurrence of active disease [112, 120].

Some studies seek to associate chemotherapeutic treatment to immunomodulatory drugs, which plays a role in therapeutic protocols by controlling clinical signs and in prevention protocols by enhancing the immune cell-mediated response through activation of macrophages via helper T cells, in order to destroy phagocytized microorganisms. Domperidone is a receptor antagonist of dopamine D2 that has been used as well. When orally given to naturally infected dogs, results showed a reduction of clinical signs and titers of anti-leishmania antibodies [121]. In Spain, Sabaté et al. [122] used a treatment protocol with domperidone in seronegative dogs in an area with high prevalence of *L. infantum* and later exposed the animals to the vector. After 21 days of treatment, the treated group had lower antibody titer, confirming that implementation of treatment with this drug reduces the risk of developing CVL [122]. Nevertheless, further clinical trials are needed to determine the optimal dose, as well as posttreatment investigations to check for parasitological cure, since the drug is a good choice for infected animals with kidney failure due to its route of administration, and it is a low-cost option [121].

Amphotericin B is a broad-spectrum macrolide antibiotic produced by actinomycete *Strepto‐ myces nodosus* [123], available for usage in VL treatment. It has a powerful leishmanicide effect and works by binding to esters on the parasite's plasma membrane [111]. According to Solano-Gallego et al., this drug has disadvantages concerning its route of administration (IV) and its nephrotoxic potential; however, its use results in less failures and recurrences [34]. Additionally, it is frequently used in the treatment of both human and canine VL for being inexpensive.

Athanasiou et al. [124] investigated the effectiveness of aminosidine sulfate, a leishmanicide antibiotic of the aminoglycoside class, in the treatment of dogs naturally infected. They observed reduction in clinical signs and parasite density, in antibody titers through indirect immunofluorescence, and in prevalence of positive dogs using PCR 3 months after the end of the experiment. These findings can be explained by direct action of the drug as well as activation of cellular immunity. However, more studies are required to prove its therapeutic efficacy for CVL, especially considering its affordable price to owners and market availability [124].

Despite the research on efficacy of different classes of medications for CVL treatment, no great progress has been done regarding toxicity or parasitological cure, highlighting the necessity for evaluation of new formulations and medicaments to be used exclusively for treatment of CVL.

## **5.2. Immunochemotherapy**

**5.1. Chemotherapy**

an undesirable option for owners.

34 Canine Medicine - Recent Topics and Advanced Research

treatment of human leishmaniasis [113].

are related to the antimony content [113].

increased parasitic load in lymph nodes [114–117].

disease [112, 120].

Due to the possibility of parasitic resistance to first-choice drugs, chemotherapeutic treatment options are limited. In addition, due to the high cost and long-term use, chemotherapy becomes

In veterinary medicine, the first choice as chemotherapy for treatment of CVL is allopurinol, a leishmaniostatic drug that acts by inhibiting leishmania growth through DNA modification. It has low cost, but parasite resistance to it remains unknown. Allopurinol is however the only drug recommended by the World Health Organization, especially since it is little used for

Antimonials are leishmanicidal drugs that hinder promastigote metabolism by inhibiting glycolytic activity. These are drugs of choice for human treatment of VL but are chemically similar to those that have been used in therapeutic protocols in dogs. Their toxicity and efficacy

When assessing the therapeutic efficacy of Glucantime alone or associated with an antigenic extract of *L. braziliensis* in experimentally infected asymptomatic dogs, reduction in parasite burden and antibody levels decreased, but actual cure was not achieved. Yet, upon using meglumine antimoniate and allopurinol—alone and in combination—in dogs naturally infected by *L. infantum*, symptomatic animals showed remission of clinical signs about 60 days after the treatment, accompanied by restoring of normal urinary and hematologic profiles. Despite clinical improvement of treated animals, however, the combination was not able to eliminate the parasite in all dogs. Thus, this protocol is not recommended, since treated dogs would go on as a source of infection for humans and other dogs, similar to results obtained using antimoniate, which promoted hematologic normalization and bone marrow recovery, but not parasitological cure, and to findings from another study wherein dogs showed

In accordance with Ikeda-Garcia et al. [116], Manna et al. [118] monitored leishmania DNA load in infected dogs through real-time PCR, using a similar treatment protocol. Therapy resulted in clinical improvement accompanied by reduction in parasite burden, but even after a long period of treatment with allopurinol alone, parasites remained in tissues [116, 118]. Miltefosine is another drug used in human treatment that has been evaluated for canine treatment in recent years. It is a phospholipid antibiotic of broad spectrum with leishmanicide effect that improves the activation of both macrophages and T cells [119]. After evaluating efficacy of three treatment protocols for dogs naturally infected with *L. infantum*, significant clinical improvement was observed and later progressed to full recovery and humoral stimulation, but did not assure cure, on the contrary, progressively increased parasite load. Hence, miltefosine alone is not recommended for treatment. The study also investigated the profile of major cytokines involved in CVL healing process, and findings showed an increase of IFN-γ within 90 days after treatment, when the parasite load in bone marrow aspirates also decreased. There was a decrease of IL-4, indicating possible resolution of the infection and efficacy of the treatment, since this cytokine is a good marker for the occurrence of active

Immunotherapy can be an effective addition to chemotherapy, as it induces effector immune response faster than the isolated use of chemotherapeutic drugs [125]. In a trial, the combination of N-methyl meglumine antimoniate (Glucantime®) and lyophilized recombinant vaccine Leish-110f®, together with adjuvant monophosphoryl lipid A plus (MPL-SE®) to treat symptomatic animals naturally infected with *L. infantum*, was effective in reducing parasitic load. This combination enables reduction of chemotherapy doses, consequently lowering the risks of toxicity and death. However, the use of multiple recombinant antigens could provide better results [126].

Borja-Cabréra et al. [127] evaluated the efficacy of immunotherapeutic vaccine Leishmune® administered alone, both in commercial formulation enriched with saponin and in laboratory formulation, compared to its use in combination with amphotericin B and allopurinol, in dogs naturally infected with *L. infantum*, but found no significant differences between the vaccine formulations tested. As for the type of treatment, the group submitted to immunochemotherapy with saponin-enriched Leishmune® vaccine abolished, not only the symptoms but also the latent infection condition, curing the dogs [127].

Joshi et al. [128] used Balb/c mice in order to verify in vivo therapeutic potential of the first generation of vaccines with dead *L. donovani* antigen (KDL) combined with adjuvant (MPL-A) and leishmanicidal chemotherapy such as cisplatin and sodium stibogluconate (SSG) and then compared those to isolated chemotherapy and immunotherapy. In animals treated with vaccine associated to SSG, there was a 98.50% reduction in serum parasitic levels, but the study also noted that the use of any of the chemotherapeutic associated with vaccination resulted in direct parasite elimination by drug activity and activation of the immune cell-mediated response. They concluded that immunochemotherapy protocols may be effective, but further studies are needed in different animal models in order to better understand the immune response [128].

## **5.3. Immunotherapy**

Immunotherapy is often considered for CVL prevention and control as a preferable alternative to euthanasia, due to the absence of a low-toxicity chemotherapy treatment and increasing resistance. According to Grandoni [129], vaccines are regarded as the best tool for eradication of the disease, particularly because it reduces the incidence of new cases, considering that the immune system fails to efficiently control the infection as it mediates a weak protective cell response. This relates to a dichotomy between the trigger of a Th1 response related to resistance and a Th2 response associated with susceptibility to infection, increased parasitic load, and strong but ineffective humoral immune response [129].

According to Joshi et al., an effective vaccine must induce a strong and long-lasting Th1 response as to prevent the initial establishment of infection: by definition, a prophylactic vaccine [128]. However, when it comes to a disease caused by an obligate intracellular protozoan, the aim is to at least control progression to severe disease and prevent transmission from host to vector, hindering maintenance of the epidemiological cycle.

So far, vaccines formulated for CVL include dead parasites, protein components or parasite subunits, purified cell fractions, vector salivary recombinant proteins, and viral particles that encode parasite's virulence factors and plasmid DNA [130]. Leishmune® (Zoetis Animal Health) was the first vaccine approved for commercial use in Brazil, in 2003. However, in 2014 the Ministry of Agriculture, Livestock, and Supply (MALS) halted manufacturing and marketing licenses due to problems in phase III. It consists of a glycoprotein antigen that binds recombinant fucose mannose which was able to stimulate good cellular immune response, decreasing IL-4, and activate CD4+ T cells, producing TNF-α and IFN-γ, important cytokines in resistance [131, 132]. Accordingly, a study done by Borja-Cabrera et al. found that the vaccine induced a long-lasting protective effect of humoral and cellular immunity, along with disappearance of clinical signs and parasitemia [133].

Leish-Tec® (Hertape), a vaccine comprising recombinant protein A2 as antigen in adjuvant saponin QuilA, continues to be marketed in Brazil since 2008, when it was recorded by the MALS. It has been demonstrated to offer partial protection against *L. infantum* in Beagle dogs, producing good humoral and cellular immune responses, high levels of anti-protein A2 IgG and IgG2, and IFN-γ [134, 135].

Despite increasing progress in production of vaccines against CVL in Brazil and worldwide, there is much to be improved regarding the induction of durable and efficient cellular and humoral immune response. Moreover, new affordable vaccines ought to be produced for the population, since those available in the market so far are expensive and not viable for use in public health.
