Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence

Abstract

Serodiagnosis of Leishmania infantum infection in dogs relies on the detection of antibodies against leishmanial crude extracts or parasitic defined antigens. The expansion of canine leishmaniasis from geographical areas of Brazil in which the infection is endemic to regions in which the disease is emerging is occurring. This fact makes necessary the analysis of the serodiagnostic capabilities of different leishmanial preparations in distinct geographical locations. In this article sera from dogs infected with Leishmania and showing the clinical form of the disease, were collected in three distinct Brazilian States and were tested against soluble leishmanial antigens or seven parasite individual antigens produced as recombinant proteins. We show that the recognition of soluble leishmanial antigens by sera from these animals was influenced by the geographical location of the infected dogs. Efficacy of the diagnosis based on this crude parasite preparation was higher in newly endemic regions when compared with areas of high disease endemicity. We also show that the use of three of the recombinant proteins, namely parasite surface kinetoplastid membrane protein of 11 kDa (KMP-11), and two members of the P protein family (P2a and P0), can improve the degree of sensitivity without adversely affecting the specificity of the diagnostic assays for canine leishmaniasis, independently of the geographical area of residence. In addition, sera from dogs clinically healthy but infected were also assayed with some of the antigen preparations. We demonstrate that the use of these proteins can help to the serodiagnosis of Leishmania infected animals with subclinical infections. Finally, we propose a diagnostic protocol using a combination of KMP-11, P2a y P0, together with total leishmanial extracts.

Keywords: Antibodies; BB, blocking buffer; CanL, Canine visceral leishmaniasis; Canine leishmaniasis; EDCB, ELISA denaturant coating buffer; ELISA, enzyme-linked immunosorbent assay; HSP, Heat shock protein; KMP-11, Kinetoplastid-membrane protein of 11 kDa; LR, Likelihood ratio; Leishmania; MS, Mato Grosso do Sul State (Brazil); PBS, phosphate saline buffer; PI, Piaui State (Brazil); ROC, Receiver Operating Characteristic; RR, Relative reactivity; RT, Room temperature; Recombinant proteins; SC, Santa Catarina State (Brazil); SLA, Soluble leishmanial antigen; Serodiagnosis; VL, Visceral leishmaniosis; WB, Washing buffer.

Graphical abstract

Fig. 1

Sera from clinically ill CanL patients collected in different geographical locations show differences in the recognition of Leishmania soluble leishmanial antigens (SLA). Sera from CanL clinically ill dogs (CI) or healthy dogs (H) collected in Santa Catarina (SC), Mato Grosso do Sul (MS) or Piaui (PI) were assayed by ELISA against soluble leishmanial antigen (SLA). In the graph it is shown the relative reactivity (RR) defined as the absorbance of a given sera divided by the absorbance of a control healthy sera included in all plates. The symbol *** indicates significant differences (P < 0.001) between clinically ill CanL and healthy sera from the same location (Mann Whitney test). The symbol +++ indicates a significant increase (P < 0.001) between the clinically ill CanL from SC with regard the equivalent sera from the other two locations and the symbol ++ indicates a significant increase in the RR values of healthy sera taken in PI with regard the equivalent sera from SC and MS (Kruskal-Wallis test) (A). Table showing the diagnostic parameters calculated by a ROC analysis (B).

Fig. 2

Reactivity of sera from clinically ill CanL patients against single Leishmania recombinant antigenic proteins. Different Leishmania antigens purified as recombinant proteins after the expression of their coding regions in E. coli were employed for coating ELISA plates: Kinetoplastid membrane protein of 11 kDa (KMP11), histone H2A, the heat shock proteins of 70 kDa (HSP70) or 83 kDa (HSP83) and the acidic ribosomal proteins P2a, P2b and P0. Sera from CanL clinically ill dogs (CI) or healthy dogs (H) collected in Santa Catarina (SC) (A), Mato Grosso do Sul (MS) (B) or Piaui (PI) (C) were assayed by ELISA. The scatter plots show the relative reactivity of the sera represented individually. Symbols * (P < 0.05), ** (P < 0.05) and *** (P < 0.001) indicate significant differences between clinically infected CanL and healthy sera from the same geographical location (Mann Whitney test).

Fig. 3

Reactivity of sera from CanL subclinically infected animals against SLA and the antigenic recombinant proteins. Sera from CanL dogs that are subclinically infected (SCI) or healthy dogs (H) were assayed by ELISA against SLA or against the indicated Leishmania antigens. A scatter plot indicating the relative reactivity values is shown. Symbols ** (P < 0.05) and *** (P < 0.001) indicate significant differences between subclinically infected CanL and healthy sera (Mann Whitney test) (A). Table showing the diagnostic parameters calculated by a ROC analysis (B).

Fig. 4

Serologic cross-reactivity of Ehrlichia canis infected dogs' sera and Leishmania antigens. Sera from E. canis infected dogs (Ec) and healthy animals (H) were assayed by ELISA against SLA or against the indicated Leishmania antigens. A scatter plot indicating the relative reactivity values is shown. Symbols * (P < 0.001) or ** (P < 0.05) indicate significant differences between samples from dogs suffering ehrlichiosis and healthy animals (Mann Whitney test).

Fig. 5

Comparative diagnostic performance of SLA and the selected leishmanial antigenic preparations. Sera from clinically ill (CI) or subclinically infected (SCI) dogs affected by CanL and dogs infected by E. canis (Ec) were assayed by ELISA against SLA or against Leishmania KMP-11, P0 and P2a antigens. A scatter plot showing the relative reactivity values (A) or a table showing the diagnostic parameters (B) from CanL clinically ill animals are shown. In (C) and (D) equivalent information for the CanL subclinically infected collection is included. Symbols ** (P < 0.05) and *** (P < 0.001) indicate significant differences between the sera from CanL animals and sera from E. canis infected dogs (Mann Whitney test).