# Diagnosis of the Multiepitope Protein rMLEEISH3 for Canine Visceral Leishmaniasis
## 1 Introduction  
Canine visceral leishmaniasisiasis (CVL) is an important zoonosis caused by _Leishmania infantum_[1], an intracellular protozoan that is transmitted mainly by bites of sand flies of the genus _Lutzomyia_, mainly of the species _Lutzomyia longiapplis_ in Brazil [2, 3, 4]. Domesticdogs are considered the main reservoir of the parasite in urban and peri-urban areas and, consequently, contribute significantly to maintaining the transmission cycle [5]. In recent decades, CVL has expanded to regions of Brazil that were not previously endemic, posing a growing challenge to public health services and veterinary surveillance agencies. Currently, CVL is endemic in at least 25 Brazilian states, with foci recorded in the North, Northeast, Southeast, and Central-West regions. According to recent data from the Brazilian Ministry of Health, more than 4000 positive cases per year have been recorded in the country, with seroprevalence rates exceeding 20% in some highly endemic municipalities [5; 6]. Control of CVL involves integrated actions, including mitigation of vector transmission, canine vaccination, and, most importantly, serological diagnosis with appropriate management of infected dogs. Although the identification of amastigote forms of the parasite in tissue samples by microscopic examination is considered an important parasitological diagnosis of leishmaniasis [7], this method has limited application in large-scale campaigns due to its invasive nature and the need for specialized personnel. The main diagnostic methods recommended by the National Leishmaniasis Surveillance and Control Program [8] for application in public campaigns are the rapid immunochromatographic test based on a dual-pathway platform (DPP(r)) and the enzyme-linked immunosorbent assay (ELISA). However, these tests have some limitations, such as low sensitivity in asymptomatic dogs, variations in immune response among animals, and possible cross-reactions with other infectious agents [9; 10]. Parasite burden plays a critical role in the progression and clinical presentation of CVL. Dogs with high parasite burdens often develop severe clinical signs and exhibit stronger humoral responses, facilitating detection by serology or PCR. In contrast, a low parasite burden--especially in asymptomatic or early stages--impairs immune activation and results in lower antibody levels, reducing the sensitivity of serological diagnostics such as ELISA. For example, [PERSON] et al. [11] found that among asymptomatic dogs with positive PCR results, nearly 18% tested negative on standard ELISA assays. Similarly, [PERSON] et al. [12] showed that dogs with low tissue parasite density exhibit immunological profiles consistent with low antibody titers and altered leukocyte patterns. These findings highlight the diagnostic challenges posed by low-parasitemic infections and justify the development of antigens capable of detecting infections in these cases. Asymptomatic dogs naturally infected with _L. infantum_ often exhibit a predominance of T-cell-mediated immune responses, with higher levels of IFN-\(\gamma\) and nitric oxide production and lower levels of IL-10 and IL-4. These responses contribute to parasite control while reducing or delaying the production of circulating antibodies, which limits the performance of conventional serological tests in detecting subclinical infections [13; 14]. This highlights the need for diagnostic antigens that can increase detection sensitivity, especially in cases of low parasite burden. In the context of serodiagnostic testing, the use of recombinant antigens is a promising alternative strategy to improve the accuracy of serological assays. Advances in bioinformatics and immunoinformatics have allowed the in silico identification of potentially immunogenic B and T epitopes, thus enabling the development of chimeric proteins that combine different antigenic regions with greater diagnostic potential [15; 16].