scholarly journals Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

2009 ◽  
Vol 16 (12) ◽  
pp. 1774-1780 ◽  
Author(s):  
Eduardo A. F. Coelho ◽  
Laura Ramírez ◽  
Mariana A. F. Costa ◽  
Vinicio T. S. Coelho ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Ribosomal Proteins ◽  
High Sensitivity ◽  
Leishmania Species ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

scholarly journals Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs

2020 ◽  
Vol 10 (3) ◽  
pp. 165-171
Author(s):  
Ingrid E. Pereira ◽  
Kyssia P. Silva ◽  
Laura M. Menegati ◽  
Aimara C. Pinheiro ◽  
Elaine A. O. Assunção ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Recombinant Proteins ◽  
Serological Test ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Reservoir Host ◽  
Superior Performance ◽  
Canine Visceral Leishmaniasis ◽  
Roc Curve Analysis

AbstractControl of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

scholarly journals Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

2013 ◽  
Vol 21 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Lourena E. Costa ◽  
Mayara I. S. Lima ◽  
Miguel A. Chávez-Fumagalli ◽  
Daniel Menezes-Souza ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Phage Display ◽  
Leishmania Infantum ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Phage Display Technology ◽  
Predictive Values ◽  
Content Type ◽  
Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

scholarly journals Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

2011 ◽  
Vol 53 (5) ◽  
pp. 283-289 ◽  
Author(s):  
Flávia Coelho Ribeiro ◽  
Armando de O. Schubach ◽  
Eliame Mouta-Confort ◽  
Tânia M.V. Pacheco ◽  
Maria de Fátima Madeira ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Large Scale ◽  
Cross Reactivity ◽  
Leishmania Braziliensis ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Canine Ehrlichiosis ◽  
Control Serum ◽  
Homologous Antigen ◽  
Heterologous Antigens

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

scholarly journals First description of autochthonous canine visceral leishmaniasis in the metropolitan region of Vitória, State of Espírito Santo, Brazil

2012 ◽  
Vol 45 (6) ◽  
pp. 754-756 ◽  
Author(s):  
Marco André Loureiro Tonini ◽  
Elenice Moreira Lemos ◽  
Alexandre Barbosa Reis ◽  
Wendel Coura Vital ◽  
Edelberto Santos Dias ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Positive Culture ◽  
Human Case ◽  
Metropolitan Region ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Leishmania Chagasi ◽  
Molecular Tests ◽  
Leishmania Spp ◽  
Polymerase Chain

INTRODUCTION: We investigated autochthonous canine visceral leishmaniasis (CVL) in the metropolitan region of Vitória (MRV), an area in which a human case was previously reported. METHODS: Serological, parasitological, and molecular tests were performed in 201 dogs. RESULTS: Twenty-six (13%) and 12 (6%) dogs were identified as positive using in-house enzyme-linked immunosorbent assay (ELISA) and rK39 tests, respectively. Two dogs had a positive culture for Leishmania chagasi, and 4 were polymerase chain reaction (PCR)-positive for Leishmania spp. One positive dog belonged to the aforementioned patient. CONCLUSIONS: Although the responsible vector was not found, our results provide evidence of autochthonous CVL in the MRV, a non-endemic area for VL.

scholarly journals An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

2009 ◽  
Vol 29 (2) ◽  
pp. 120-124 ◽  
Author(s):  
Débora Carvalho ◽  
Trícia M.F.S. Oliveira ◽  
Cristiane D. Baldani ◽  
Rosangela Z. Machado
Keyword(s):  
Visceral Leishmaniasis ◽  
Immunosorbent Assay ◽  
Fluorescent Antibody ◽  
Domestic Dog ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Specificity And Sensitivity ◽  
Belo Horizonte

Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.

scholarly journals Evaluation of Recombinant Antigens for Serodiagnosis of Chagas’ Disease in South and Central America

1999 ◽  
Vol 37 (5) ◽  
pp. 1554-1560 ◽  
Author(s):  
Eufrosina S. Umezawa ◽  
Sueli F. Bastos ◽  
Mario E. Camargo ◽  
Luci M. Yamauchi ◽  
Márcia R. Santos ◽  
...  
Keyword(s):  
Chagas Disease ◽  
Recombinant Proteins ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Recombinant Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serological Tests ◽  
Recombinant Antigens ◽  
Fusion Products

The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions ofTrypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzirecombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.

scholarly journals Multicomponent Chimeric Antigen for Serodiagnosis of Canine Visceral Leishmaniasis

1998 ◽  
Vol 36 (1) ◽  
pp. 58-63 ◽  
Author(s):  
Manuel Soto ◽  
Jose M. Requena ◽  
Luis Quijada ◽  
Carlos Alonso
Keyword(s):  
Visceral Leishmaniasis ◽  
Bacterial Production ◽  
Ribosomal Proteins ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Histone H2a ◽  
Test Elisa ◽  
Gene Coding ◽  
High Level

In this work, we describe the assembly of a synthetic gene coding for several antigenic determinants found in different Leishmania infantum antigens. Selected epitopes were derived from the ribosomal proteins LiP2a, LiP2b, and LiP0 and from the histone H2A. The resulting gene was overexpressed in Escherichia coli either as a fusion protein (with the vector pMAL-c2) or alone (with the vector pQE). In both cases, high-level bacterial production of the recombinant protein was achieved and the products were found to be stable. Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments confirmed that the corresponding epitopes are present in the engineered protein. Finally, a serological evaluation of this multiple-epitope protein by Falcon assay screening test-ELISA revealed a sensitivity of 79 to 93% and a specificity of 96 to 100% in diagnosis of canine visceral leishmaniasis, indicating that this protein represents a valuable tool for serodiagnosis.

scholarly journals Canine visceral leishmaniasis and Chagas disease among dogs in Araguaína, Tocantins

2013 ◽  
Vol 22 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Arielle Nunes Morais ◽  
Marlos Gonçalves Sousa ◽  
Luciana Regina Meireles ◽  
Norival Kesper Jr. ◽  
Eufrosina Setsu Umezawa
Keyword(s):  
Visceral Leishmaniasis ◽  
Sao Paulo ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
São Paulo ◽  
Reference Test ◽  
Serum Samples ◽  
Canine Population ◽  
De Medicina

The present study analyzed serum samples from 111 male and female dogs of various ages from the municipality of Araguaína in the State of Tocantins, Brazil. Serological diagnosis of canine visceral leishmaniasis (CVL) was initially performed at the Central Laboratory (Laboratório Central – LACEN) of Araguaína, resulting in 61 positive samples by an indirect immunofluorescence assay (IIFA) (≥1:40) and 50 non-reactive samples. The same samples were analyzed at the São Paulo Institute of Tropical Medicine (Instituto de Medicina Tropical de São Paulo – IMTSP) by an enzyme-linked-immunosorbent assay (ELISA), resulting in 57 positive samples (51.35%) and 54 negative samples (48.64%). The Kappa coefficient of agreement between the tests was 0.74. The serum samples were also subjected to a diagnostic assay for Trypanosoma cruzi(Trypomastigote Excreted/Secreted Antigens -TESA-blot) that detected five suspect animals; three of those animals were positive for leishmaniasis by ELISA but negative by IIFA. These findings suggest that the canine population of Araguaína may be simultaneously infected withLeishmania chagasi and T. cruzi. The results obtained demonstrate the difficulty of using serology to detect CVL, thus emphasizing the necessity for a reference test to diagnose CVL, particularly in regions where the infection is endemic.

scholarly journals trans-Sialidase Neutralizing Antibody Detection in Trypanosoma cruzi-Infected Domestic Reservoirs

2011 ◽  
Vol 18 (6) ◽  
pp. 984-989 ◽  
Author(s):  
Paula A. Sartor ◽  
Martha V. Cardinal ◽  
Marcela M. Orozco ◽  
Ricardo E. Gürtler ◽  
M. Susana Leguizamón
Keyword(s):  
Visceral Leishmaniasis ◽  
Trypanosoma Cruzi ◽  
Neutralizing Antibodies ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Trypanosoma Rangeli ◽  
Serum Samples ◽  
Dipstick Test ◽  
Content Type

ABSTRACTThe detection ofTrypanosoma cruziinfection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected withLeishmaniaspp. We used atrans-sialidase inhibition assay (TIA) forT. cruzidiagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinanttrans-sialidase, an enzyme that is not detected in the coendemicLeishmaniaspecies orTrypanosoma rangeliparasites.T. cruziinfection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development oftrans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence oftrans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool forT. cruzidetection in the main domestic reservoirs.

scholarly journals Dogs as Sentinels for Flavivirus Exposure in Urban, Peri-Urban and Rural Hanoi, Vietnam

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 507
Author(s):  
Long Pham-Thanh ◽  
Thang Nguyen-Tien ◽  
Ulf Magnusson ◽  
Vuong Bui-Nghia ◽  
Anh Bui-Ngoc ◽  
...  
Keyword(s):  
Mosquito Control ◽  
Risk Mitigation ◽  
Significant Risk ◽  
Cross Sectional Study ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Cross Sectional ◽  
Major Health ◽  
Household Level

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.

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