**6. Diagnosis**

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

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

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

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

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

latent infection condition, curing the dogs [127].

36 Canine Medicine - Recent Topics and Advanced Research

strong but ineffective humoral immune response [129].

from host to vector, hindering maintenance of the epidemiological cycle.

response [128].

**5.3. Immunotherapy**

The clinical diagnosis of CVL is challenging, as signs are usually not specific for the disease; laboratory assays are therefore of paramount importance. Moreover, as the dog is considered to be the major reservoir in Brazil, serological assays constitute the basis to identify infected dogs and to direct public health actions aiming the disease control.

The indirect immunofluorescence antibody test (IFAT) was established as the standard serodiagnosis in public health programs more than 40 years ago. It was later substituted by an ELISA based on crude leishmania antigens [111] and more recently by a recombinant ELISA and an immunochromatographic rapid test for detecting K26/K39-reactive antibodies in canine sera [136, 137]. According to the recommendation from the Brazilian Ministry of Health, sera collected from dogs in seroepidemiologic surveys as part of the Leishmaniasis Control Program are first screened using the fast immunochromatographic assay (known as dual-path platform (DPP)), and the positive samples are retested in the recombinant ELISA. The approach undoubtedly speeds up the screening [138], but due to the DPP low sensitivity in cases of sera from asymptomatic dogs [137, 139, 140], a sizable set of infected dogs possibly remains in the endemic areas, jeopardizing the effectiveness of control actions.

Private laboratories used to rely in a single result obtained by the use of a commercial recombinant ELISA kit (ELISA S7) registered at the Brazilian Ministry of Agriculture, Livestock, and Supply for the diagnosis of CVL (www.biogene.ind.br). More recently, some fast immunochromatographic assays started to be used, such as the Alere assay [141], but high costs preclude their adoption in the routine diagnosis. A recombinant K39-based ELISA developed for research use only (http://www.inbios.com/kalazar-detect-elisa-system-for-visceralleishmaniasis-intl/) has also seen some use in routine commercial CVL diagnosis.

The official recombinant ELISA assay and the ELISA S7 have similar sensitivity and specificity indexes and perform equally well in the identification of seropositive, supposedly infected dogs. They also do not display significant rates of positive reactions in cases of vaccinated dogs. Other serological assays, e.g., the direct agglutination (DAT), are not easily available in Brazil and were never adopted in private or public labs. Although new antigens have been described in the last years (e.g., [142]), their commercial use in serodiagnosis is still uncertain. Although available as commercial kits (http://www.genesig.com/products/ 9332?gclid=CNXfgtXc\_c4CFcoHkQodMm4Ctw), PCR assays are seldom used and have limited application for the routine diagnosis.

As the existing recombinant ELISA assays have high sensitivity and specificity even in the serodiagnosis of CVL in asymptomatic dogs, one could argue against the need of new laboratory tests. However, claims of cross reactions with babesiosis and other common canine infectious diseases [138] continue to stir dissatisfaction.

In conclusion, no diagnostic breakthroughs have been described and no innovative technology was introduced in the market in the last years, and there are no evidences that this scenario will be changed in the near future.
