Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples
Abstract Background Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isother...
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| Format: | Article |
| Language: | English |
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BMC
2015-07-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-015-0991-2 |
| _version_ | 1827933866892984320 |
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| author | Chun-hua Gao Dan Ding Jun-yun Wang Dietmar Steverding Xia Wang Yue-tao Yang Feng Shi |
| author_facet | Chun-hua Gao Dan Ding Jun-yun Wang Dietmar Steverding Xia Wang Yue-tao Yang Feng Shi |
| author_sort | Chun-hua Gao |
| collection | DOAJ |
| description | Abstract Background Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3 %, 58.6 %, 40.5 % and 10.8 % by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97 % for both methods. Conclusion This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China. |
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| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-13T07:30:39Z |
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| spelling | doaj.art-8481b649ed4a43cf8615544e100300272023-06-04T11:08:12ZengBMCParasites & Vectors1756-33052015-07-01811810.1186/s13071-015-0991-2Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samplesChun-hua Gao0Dan Ding1Jun-yun Wang2Dietmar Steverding3Xia Wang4Yue-tao Yang5Feng Shi6National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Center for International Research on Tropical Diseases, WHO Collaborating Centre for Malaria, Schistosomiasis and FilariasisNational Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Center for International Research on Tropical Diseases, WHO Collaborating Centre for Malaria, Schistosomiasis and FilariasisNational Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Center for International Research on Tropical Diseases, WHO Collaborating Centre for Malaria, Schistosomiasis and FilariasisBioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East AngliaBioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East AngliaNational Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Center for International Research on Tropical Diseases, WHO Collaborating Centre for Malaria, Schistosomiasis and FilariasisNational Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Center for International Research on Tropical Diseases, WHO Collaborating Centre for Malaria, Schistosomiasis and FilariasisAbstract Background Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3 %, 58.6 %, 40.5 % and 10.8 % by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97 % for both methods. Conclusion This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China.https://doi.org/10.1186/s13071-015-0991-2Loop-mediated isothermal amplificationConjunctional swabsZoonotic visceral leishmaniasisAsymptomatic canine reservoir hostLeishmania infantum |
| spellingShingle | Chun-hua Gao Dan Ding Jun-yun Wang Dietmar Steverding Xia Wang Yue-tao Yang Feng Shi Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples Parasites & Vectors Loop-mediated isothermal amplification Conjunctional swabs Zoonotic visceral leishmaniasis Asymptomatic canine reservoir host Leishmania infantum |
| title | Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples |
| title_full | Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples |
| title_fullStr | Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples |
| title_full_unstemmed | Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples |
| title_short | Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples |
| title_sort | development of a lamp assay for detection of leishmania infantum infection in dogs using conjunctival swab samples |
| topic | Loop-mediated isothermal amplification Conjunctional swabs Zoonotic visceral leishmaniasis Asymptomatic canine reservoir host Leishmania infantum |
| url | https://doi.org/10.1186/s13071-015-0991-2 |
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