Prokaryotic expression and antigenic characterization of three recombinant Leishmania antigens for serological diagnosis of canine leishmaniasis.
Three recombinant antigens of Leishmania chagasi (= L. infantum) were expressed in prokaryotic systems and evaluated (using a panel of dog sera characterized by parasitological and serological immunofluorescent antibody test [IFAT] techniques) as diagnostic markers of infection. The whole open reading frame encoding K9, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment encoding a single 39-amino-acid subunit of the kinesin-related protein K39 (K39sub) were amplified from L. infantum DNA and cloned into a pGEX-2T expression vector in frame with glutathione S-transferase (GST). The sensitivity and specificity of enzyme-linked immunosorbent assays (ELISAs) using K26 as an antigen (evaluated with sera from 20 parasitologically positive and 20 parasitologically negative dogs) were both 100% (95% confidence interval [CI] = 83.2 to 100). When K9 and K39sub were used, sensitivity was 95% (95% CI = 75.1 to 99.9) and specificity was 100% (95% CI = 83.2 to 100). Using 182 field sera, a good agreement was found between the recombinant K26 ELISA and IFAT (K = 0.92; 95% CI = 0.86 to 0.98) results and between the K9 and K39sub ELISA (used in parallel) and IFAT (K = 0.87; 95% CI = 0.80 to 0.95) results. The results demonstrate that each antigen carries immunodominant epitopes and that their combination may further increase the sensitivity of currently available serological tests.
Rosati
S
S
Dipartimento di Produzioni Animali, Epidemiologia ed Ecologia, Università di Torino, 10095 Grugliasco (TO), Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, Sezione di Imperia, 18100 Imperia, Italy. sergio.rosati@unito.it
Ortoffi
M
M
Profiti
M
M
Mannelli
A
A
Mignone
W
W
Bollo
E
E
Gradoni
L
L
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D003001:Cloning, Molecular; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D016056:Immunodominant Epitopes; D018314:Leishmania infantum / Q000235:genetics / Q000276:immunology*; D007896:Leishmaniasis / Q000175:diagnosis*; D011387:Prokaryotic Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods
D000953:Antigens, Protozoan; D016056:Immunodominant Epitopes; D011994:Recombinant Proteins