Prokaryotic Expression and Antigenic Characterization of Three Recombinant<i>Leishmania</i>Antigens for Serological Diagnosis of Canine Leishmaniasis

Rosati, Sergio; Ortoffi, Marco Francesco; Profiti, Margherita; Mannelli, Alessandro; Mignone, Walter; Bollo, E.; Gradoni, Luigi

doi:10.1128/cdli.10.6.1153-1156.2003

Cited by 31 publications

(36 citation statements)

References 12 publications

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“…The difference in sensitivity might be that a quantitative method was used in the current study whereas the rK26 strip test used in the other study is a qualitative method of antibody detection. Other studies conducted in symptomatic canine VL cases in Brazil showed higher sensitivity by ELISA using rK9 and rK26 antigen [20][21][22]. The lower sensitivity in this study may be due to the fact that dog is a different species than human.…”

Section: Discussioncontrasting

confidence: 51%

Compararative evaluation of rK9, rK26 and rK39 antigens in the serodiagnosis of Indian visceral leishmaniasis

Mohapatra¹,

Singh

Sen

et al. 2009

J Infect Dev Ctries

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Background: This study was designed for comparative evaluation of two relatively newer recombinant hydrophilic antigens, rK9 and rK26 of Leishmania chagasi along with rK39 (a 39-aminoacid-repetitive immunodominant B-cell epitope of kinesin-related antigen from L. chagasi) and crude soluble antigen (CSA) for the serodiagnosis of Indian visceral leishmaniasis (VL) patients by quantitative ELISA. Methodology: In the present study a total of 80 subjects comprising of 55 confirmed VL cases and 25 endemic controls (EC) were subjected to ELISA using four different antigens, namely rK9, rK26, rK39 and CSA (derived from Leishmania donovani promastigotes). Results: Sensitivity was as follows: 78% (95%CI 63-100%) for rK9, 38% (95%CI 28-59%) for rK26, 100% for rK39, and 80% (95% CI 65-100%) for CSA. The specificity of rK9, rK26, rK39 and CSA was found to be 84% (95%CI 61-100%), 80% (95%CI 56-100%), 96% (95%CI 75-100%) and 72% (95%CI 49-100%), respectively. Conclusions: rK39 was observed to be the most suitable antigen as compared to rK26 and rK9 whereas rK9 performed better than rK26. Hence rK9 antigen may either be used as an adjunct to rK39 for accurate diagnosis of VL or may be used in the absence or non-availability of rK39 antigen for the serodiagnosis.

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Section: Discussioncontrasting

confidence: 51%

Compararative evaluation of rK9, rK26 and rK39 antigens in the serodiagnosis of Indian visceral leishmaniasis

Mohapatra¹,

Singh

Sen

et al. 2009

J Infect Dev Ctries

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“…Two recombinant plasmids containing, respectively, the whole open reading frame of K9 antigen (GenBank accession number AF131227) and the K39sub (GenBank accession number L07879) have been developed previously (34) and served as PCR templates in this study.…”

Section: Figmentioning

confidence: 99%

“…The three recombinant antigens showed independent and complementary immunoreactivities and reached an excellent agreement with IFAT when used in parallel (34). An ideal test would therefore employ a combination of relevant epitopes in a single recombinant antigen, more specific than crude antigen preparation and more sensitive than single epitope-based ELISA.…”

mentioning

confidence: 99%

Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes ofLeishmania infantumfor Serodiagnosis of Visceral Leishmaniasis

Boarino

Scalone

Gradoni

et al. 2005

Clin Vaccine Immunol

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Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused byLeishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n ‫؍‬ 180) and canine (n ‫؍‬ 343) control groups, while sensitivity was higher in canine VL (96%, n ‫؍‬ 213) than in human VL (82%, n ‫؍‬ 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k ‫؍‬ 0.95, 95% confidence interval ‫؍‬ 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k ‫؍‬ 0.81, 95% confidence interval ‫؍‬ 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts. Animal and human leishmaniases are parasitic infections caused by protozoan hemoflagellates belonging to the genus Leishmania.Parasites are transmitted by the bite of phlebotomine sand flies to the mononuclear phagocyte system of the vertebrate host, where the infecting promastigotes differentiate into and replicate as amastigotes. The geographical distribution and the spreading of the infection depend on the presence of sand fly vectors and of animal reservoirs (3).Wild canids and domestic dogs represent the main reservoir hosts of zoonotic visceral leishmaniasis (VL), playing a strategic role for diffusion and maintenance of the infection (25). Zoonotic VL is caused by Leishmania infantum (syn. Leishmania chagasi) (24) and spread in the Mediterranean basin, in the Middle East, and in Latin America.In the past decades, human factors and environmental changes have promoted the diffusion of the disease in areas originally not considered suitable for the spreading of leishmaniasis (9,11,32,...

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“…HSP70 is a target for the humoral response during human leishmaniasis (13) and has been shown to stimulate the murine immune system (23). rK39 is mainly expressed in the amastigote stage and elicits a strong immune response in both asymptomatic and clinically infected dogs (26).…”

mentioning

confidence: 99%

Kinetics and Diagnostic and Prognostic Potential of Quantitative Western Blot Analysis and Antigen-Specific Enzyme-Linked Immunosorbent Assay in Experimental Canine Leishmaniasis

Talmi-Frank

Strauss-Ayali

Jaffe

et al. 2006

Clin Vaccine Immunol

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Quantitative computerized Western blot analysis of antibody responses during experimental canine Leishmania infantum infection distinguished between immunodominant and nonimmunodominant protein bands. Six infected beagles, positive by both PCR and parasite culture, were monitored over 75 weeks postinfection and during a 12-week allopurinol treatment course. All dogs were symptomatic at the time of treatment. Of 12 antigenic bands examined, the immunodominant bands (12, 14, 24, 29, 48, and 68 kDa) showed significantly increased intensities (P < 0.01) and higher frequencies of recognition than the nonimmunodominant bands at all time points. Detection of the former bands at 6 weeks postinfection preceded seroconversion by enzymelinked immunosorbent assay (ELISA) both on crude Leishmania antigen or the recombinant proteins rK39 and HSP70. Reactivity with the 14-, 48-, and 68-kDa bands signified early infection, whereas increased reactivity with the 14-, 24-, and 29-kDa bands was associated with posttreatment parasite persistence and potential unfavorable prognosis. Total lane intensity (TLI) emerged as a sensitive marker for early infection and increased as early as 4 weeks postinfection. TLI had a significantly higher (P < 0.01) relative increase rate than crude Leishmania antigen or HSP70 or rK39 ELISA at all time points. These immunodominant antigens and TLI, as determined by quantitative Western blotting, will be valuable for early detection and treatment evaluation of canine leishmaniasis.

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Prokaryotic Expression and Antigenic Characterization of Three RecombinantLeishmaniaAntigens for Serological Diagnosis of Canine Leishmaniasis

Cited by 31 publications

References 12 publications

Compararative evaluation of rK9, rK26 and rK39 antigens in the serodiagnosis of Indian visceral leishmaniasis

Compararative evaluation of rK9, rK26 and rK39 antigens in the serodiagnosis of Indian visceral leishmaniasis

Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes ofLeishmania infantumfor Serodiagnosis of Visceral Leishmaniasis

Kinetics and Diagnostic and Prognostic Potential of Quantitative Western Blot Analysis and Antigen-Specific Enzyme-Linked Immunosorbent Assay in Experimental Canine Leishmaniasis

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