Abstract :Canine Visceral Leishmaniasis (CVL) is an infection of veterinary importance, involving a
complex interaction between trypanosomatid protozoa of the Leishmania donovani complex, sand
fly vectors, environmental conditions that influence the distribution of the vector, dogs that are the
major urban reservoirs and humans that are hosts that harbor these parasites. Dogs are a key piece
in the epidemiological chain of VL because it has been shown that the high prevalence of canine
infection is related to a high risk of disease in humans. Detection of the infection in dogs is essential
to proceed to the chemotherapeutic treatment in infected individuals, in epidemiological studies
and control programs, for this reason, the implementation of rapid, sensitive and specific diagnostic
tests is essential. The diagnosis of CVL commonly uses extracts from the promastigote stage of
Leishmania, which are complex and heterogeneous mixtures that may have values of low specificity
(false positives) and percentages of minimal sensitivity (false negatives). In order to achieve the
improvement of diagnostic tests, numerous investigations based on Molecular Biology techniques
have been carried out over several years, which have allowed the obtaining of recombinant antigens
from Leishmania donovani complex cDNA libraries. In this way excellent antigens have been
obtained, such as rk39 and rk28, which are very specific, sensitive, obtained in large quantities at
a relatively low cost through an easy and fast purification process. The recombinant protein rK39
is a 39 amino acid repetitive immunodominant B cell epitope that is part of a 230 kDa kinesinrelated
protein expressed predominantly in the amastigotes of viscerotropic L. chagasi, has proved
to be an exceptionally strong marker of disease, allows the detection of canine VL in asymptomatic
animals, as well as in those that present clinical symptoms. The levels of anti-rK39 antibodies are
highly correlated with active disease can be used to monitor the effectiveness of chemotherapeutic
compounds and clinical monitoring of patients. The incorporation of this antigen in a dipstick
format allows the production of a Rapid Diagnostic Test (RDT), easy to use, which provides reliable
results, without the need for equipment or specialized personnel, all these advantages allow its
implementation in the field; this is very important for handling in rural areas where humans and
dogs of low socioeconomic conditions live. Subsequent studios have allowed to express rK9 and
rK26, two related hydrophilic antigens of L. chagasi that differ for the presence of 11 copies of a
14-amino-acid repeat in the open reading frame of K26. Some evaluations have shown that the
sensitivity of the rK26 antigen is only 20% to 40% while rK9 yields 78% sensitivity. The antigenicity
of K9, K39, and K26 was determined in multiple-well ELISA using infected dog sera, these antigens
showed independent and complementary immunoreactivities, which led to the idea of producing
an antigen formed by the fusion of said proteins. Thus to solve these drawbacks, the technological
advances of the last few years have been used to develop new generation of recombinant chimeric
protein, resulting from fusion of L. infantum genes: k9, k39 and k26, denominated rK28 chimeric
protein with multiple tandem repeat sequences, increasing antigen epitope density, with a sensitivity
of 92% to 100% in Sudan. Due to the multiple advantages of rK28 has been used in the development
of RDT, for example, DPP CVL a screening method established by the Brazilian government. this
test shows many benefits: i) high levels of sensitivity and specificity for canine VL, ii) shows results
in 15 minutes, which allows to evaluate a large number of samples, iii) in association with the
confirmatory test EIE CVL give results within 15 days. Their incorporation in the current protocol
accelerates the implementation of the control measures in endemic areas endemics. In conclusion,
this assay constitute an approximation to the ideal test because it employs a combination of relevant
epitopes in a single recombinant antigen in the form of quimera, more specific than crude antigen
preparation and more sensitive than single epitope-based ELISA.