scholarly journals Evaluation of a pan-Leishmania SL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

2020 ◽  
Author(s):  
Myrthe Pareyn ◽  
Rik Hendrickx ◽  
Nigatu Girma ◽  
Sarah Hendrickx ◽  
Lieselotte Van Bockstal ◽  
...  
Keyword(s):  
Low Cost ◽  
Qpcr Assay ◽  
Sand Fly ◽  
Sand Flies ◽  
Kinetoplast Dna ◽  
Rna Detection ◽  
Tissue Samples ◽  
Spliced Leader ◽  
Reservoir Hosts

AbstractBackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR Green quantitative PCR (qPCR) assay which specifically detects the conserved spliced-leader RNA (SL-RNA) sequence has recently been developed. This study comparatively assessed the SL-RNA assay performance for the detection of Leishmania in field and laboratory infected sand flies and in tissue samples from hyraxes as reservoir hosts.Principal findingsThe qPCRs targeting SL-RNA and kDNA performed equally well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL-RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL-RNA levels were approximately 3-fold lower in sand fly promastigotes (ΔCt 1.7). The theoretical limit of detection and quantification of the SL-RNA qPCR respectively reached down to 10−3 and 10 parasite equivalents. SL-RNA detection in stored hyrax samples was less efficient with some false negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionThis study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay provides complementary information to the standard kDNA assays, since it is pan-Leishmania specific and detects viable parasites, a prerequisite for identification of vectors and reservoirs.Author summaryIn order to identify vectors and reservoirs of Leishmania, a large number of sand fly and animal tissue samples needs to be screened, because the infection prevalence is generally low. Hence, sensitive low-cost methods are required for nucleic acid isolation and Leishmania detection. Most approaches amplify DNA targets, in particular minicircle kinetoplast DNA (kDNA). Recently, a qPCR was developed that detects the spliced-leader RNA (SL-RNA) sequence, which is conserved among various Leishmania species and allows detection of viable parasites. We show that the SL-RNA qPCR is highly compatible with a low-cost, crude extraction approach and performs equally well on laboratory and field infected sand fly samples as kDNA qPCR assays. The assay can detect 10−3 parasite equivalent in sand flies and enables Leishmania quantification down to 10 parasites. We found that the copy number of SL-RNA is 3-fold lower in sand fly derived promastigotes compared to cultured promastigotes. SL-RNA detection in hyrax tissue samples appeared less efficient, which is presumably due to long-term storage without RNA stabilizing reagents. Overall, our assay is complementary to kDNA assays as it can identify viable Leishmania stages, which provides pivotal information for identification of reservoirs and vectors and their transmission capacity.

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2019 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Post Inoculation ◽  
Sand Flies ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Reservoir Hosts ◽  
Lutzomyia Migonei

Abstract BackgroundLeishmaniasis is a human and animal disease caused by parasites of the genus Leishmania, which is now divided into 4 subgenera – L. (Leishmania), L. (Viannia), L. (Sauroleishmania) and L. (Mundinia). Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis, L. orientalis and unnamed L. sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. MethodsExperimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 107 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weakly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei. The distribution of parasites in different tissues was determined post mortem by conventional PCR.ResultsNo significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L. sp. from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation.ConclusionsAccording to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.

Influence of Deforestation on the Community Structure of Sand Flies (Diptera: Psychodidae) in Eastern Amazonia

2019 ◽  
Vol 56 (4) ◽  
pp. 1004-1012
Author(s):  
José Manuel Macário Rebêlo ◽  
Jorge Luiz Pinto Moraes ◽  
Gustavo Barbosa Vieira Cruz ◽  
Joudellys Andrade-Silva ◽  
Maria Da Conceição Abreu Bandeira ◽  
...  
Keyword(s):  
Community Structure ◽  
Species Composition ◽  
Significant Influence ◽  
Sand Fly ◽  
Forest Fragments ◽  
Sand Flies ◽  
Human Occupation ◽  
Short Term

Abstract Variation in the structure of phlebotomine (sand fly) communities in forest fragments with different degrees of preservation and human occupation (peridomicile) in eastern Amazonia was studied. We identified 43 species of sand flies in our study, of which 38 occurred in both preserved forest areas and in the peridomiciles of short-term settlements, while another 28 species occurred in altered forest fragments and long-term settlements. The composition of the community at each site changed with the type of environment (forest or peridomicile), with the species Lutzomyia evandroi, L. whitmani, L. choti, L. serrana, L. triacantha, L. migonei, L. hirsuta, L. shannoni, and L. brachyphylla accounting for more than 54% of the differences among environments. The quality of the environment exerted a significant influence on the structure of phlebotomine communities, and affected their species composition, richness, and abundance.

scholarly journals Molecular detection of Leishmania donovani, Leishmania major, and Trypanosoma species in Sergentomyia squamipleuris sand flies from a visceral leishmaniasis focus in Merti sub-County, eastern Kenya

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Barrack O. Owino ◽  
Jackline Milkah Mwangi ◽  
Steve Kiplagat ◽  
Hannah Njiriku Mwangi ◽  
Johnstone M. Ingonga ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Mus Musculus ◽  
Blood Meal ◽  
Leishmania Donovani ◽  
Leishmania Major ◽  
Sand Fly ◽  
Health Concern ◽  
Sand Flies ◽  
Reservoir Hosts ◽  
Blood Meals

Abstract Background Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts. Methods We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. Results We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. Conclusions Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2020 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Sand Flies ◽  
Animal Disease ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Reservoir Hosts ◽  
Lutzomyia Migonei

Abstract Background: Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania, which is now divided into 4 subgenera – L. (Leishmania), L. (Viannia), L. (Sauroleishmania) and L. (Mundinia). Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis, L. orientalis and unnamed L. sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. Methods: Experimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 107 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei. The distribution of parasites in different tissues was determined post mortem by conventional PCR.Results: No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L. sp. from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation.Conclusions: According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.

scholarly journals A Listeria monocytogenes-Based Vaccine That Secretes Sand Fly Salivary Protein LJM11 Confers Long-Term Protection against Vector-Transmitted Leishmania major

2014 ◽  
Vol 82 (7) ◽  
pp. 2736-2745 ◽  
Author(s):  
Delbert S. Abi Abdallah ◽  
Alan Pavinski Bitar ◽  
Fabiano Oliveira ◽  
Claudio Meneses ◽  
Justin J. Park ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Cutaneous Leishmaniasis ◽  
Leishmania Major ◽  
Salivary Protein ◽  
Sand Fly ◽  
Sand Flies ◽  
Content Type ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACTCutaneous leishmaniasis is a sand fly-transmitted disease characterized by skin ulcers that carry significant scarring and social stigmatization. Over the past years, there has been cumulative evidence that immunity to specific sand fly salivary proteins confers a significant level of protection against leishmaniasis. In this study, we used an attenuated strain ofListeria monocytogenesas a vaccine expression system for LJM11, a sand fly salivary protein identified as a good vaccine candidate. We observed that mice were best protected against an intradermal needle challenge withLeishmania majorand sand fly saliva when vaccinated intravenously. However, this protection was short-lived. Importantly, groups of vaccinated mice were protected long term when challenged with infected sand flies. Protection correlated with smaller lesion size, fewer scars, and better parasite control between 2 and 6 weeks postchallenge compared to the control group of mice vaccinated with the parentL. monocytogenesstrain not expressing LJM11. Moreover, protection correlated with high numbers of CD4+, gamma interferon-positive (IFN-γ+), tumor necrosis factor alpha-positive/negative (TNF-α+/−), interleukin-10-negative (IL-10−) cells and low numbers of CD4+IFN-γ+/−TNF-α−IL-10+T cells at 2 weeks postchallenge. Overall, our data indicate that delivery of LJM11 byListeriais a promising vaccination strategy against cutaneous leishmaniasis inducing long-term protection against ulcer formation following a natural challenge with infected sand flies.

scholarly journals Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Myrthe Pareyn ◽  
Rik Hendrickx ◽  
Nigatu Girma ◽  
Sarah Hendrickx ◽  
Lieselotte Van Bockstal ◽  
...  
Keyword(s):  
Qpcr Assay ◽  
Sand Flies ◽  
Parasite Detection ◽  
Reservoir Hosts

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2020 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Post Inoculation ◽  
Sand Flies ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Reservoir Hosts ◽  
Lutzomyia Migonei

Abstract Background: Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania , which is now divided into 4 subgenera – L. (Leishmania) , L. (Viannia) , L. (Sauroleishmania) and L. (Mundinia) . Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis , L. orientalis and unnamed L . sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. Methods: Experimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 10 7 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei . The distribution of parasites in different tissues was determined post mortem by conventional PCR. Results: No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L . sp . from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation. Conclusions: According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2020 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Post Inoculation ◽  
Sand Flies ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Leishmania Enriettii ◽  
Reservoir Hosts

Abstract Background: Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania , which is now divided into 4 subgenera – L. (Leishmania) , L. (Viannia) , L. (Sauroleishmania) and L. (Mundinia) . Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis , L. orientalis and unnamed L . sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. Methods: Experimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 10 7 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei . The distribution of parasites in different tissues was determined post mortem by conventional PCR. Results: No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L . sp . from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation. Conclusions: According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms. Key words: Leishmania, Mundinia , guinea pig, Leishmania enriettii, Leishmania martiniquensis, Leishmania orientalis, Leishmania macropodum, animal model

Fine Particle Mass Monitoring with Low-Cost Sensors: Corrections and Long-Term Performance Evaluation

2019 ◽  
Author(s):  
Carl Malings ◽  
Rebecca Tanzer ◽  
Aliaksei Hauryliuk ◽  
Provat K. Saha ◽  
Allen L. Robinson ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Fine Particle ◽  
Low Cost ◽  
Particle Mass ◽  
Long Term Performance ◽  
Term Performance

scholarly journals Wittichenite semiconductor of Cu3BiS3 films for efficient hydrogen evolution from solar driven photoelectrochemical water splitting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dingwang Huang ◽  
Lintao Li ◽  
Kang Wang ◽  
Yan Li ◽  
Kuang Feng ◽  
...  
Keyword(s):  
Hydrogen Evolution ◽  
Water Splitting ◽  
Low Cost ◽  
Solar Hydrogen ◽  
Onset Potential ◽  
Term Stability ◽  
Long Term Stability ◽  
Solar Water Splitting ◽  
Current Densities

AbstractA highly efficient, low-cost and environmentally friendly photocathode with long-term stability is the goal of practical solar hydrogen evolution applications. Here, we found that the Cu3BiS3 film-based photocathode meets the abovementioned requirements. The Cu3BiS3-based photocathode presents a remarkable onset potential over 0.9 VRHE with excellent photoelectrochemical current densities (~7 mA/cm2 under 0 VRHE) and appreciable 10-hour long-term stability in neutral water solutions. This high onset potential of the Cu3BiS3-based photocathode directly results in a good unbiased operating photocurrent of ~1.6 mA/cm2 assisted by the BiVO4 photoanode. A tandem device of Cu3BiS3-BiVO4 with an unbiased solar-to-hydrogen conversion efficiency of 2.04% is presented. This tandem device also presents high stability over 20 hours. Ultimately, a 5 × 5 cm2 large Cu3BiS3-BiVO4 tandem device module is fabricated for standalone overall solar water splitting with a long-term stability of 60 hours.

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