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Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea.

ABSTRACT: Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2?c-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts.

SUBMITTER: Ndzeshang LB

PROVIDER: S-EPMC7470323 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea.

Ndzeshang Lontum B LB Fombad Fanny F FF Njouendou Abdel J AJ Chunda Valerine C VC Gandjui Narcisse V T NVT Akumtoh Desmond N DN Chounna Patrick W N PWN Steven Andrew A Pionnier Nicolas P NP Layland Laura E LE Ritter Manuel M Hoerauf Achim A Taylor Mark J MJ Turner Joseph D JD Wanji Samuel S

PLoS neglected tropical diseases 20200817 8

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. sila ...[more]

PMID: 32804951

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Project description:BackgroundThe mass drug administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that has, in turn, led to reduction of infection levels in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission as it provides a potential alternative or indirect strategy for addressing the problem of co-endemic loiasis and lymphatic filariasis through the Onchocerciasis Elimination Programme and it further reflects the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, the sensitivity of this method is compromised when the intensity of infection is low. The loop-mediated isothermal amplification (LAMP) assay of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. The aim of this study was to validate the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions.MethodsTwo sets of 18 flies were fed on volunteers with either a low (< 10 mf/ml) or high (> 30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed using LAMP for the detection of L. loa infection. In addition, a total of 9270 flies were collected from the north-west, east, and south-west regions (SW 1 and 2) of Cameroon using sweep nets and subjected to microscopy (7841 flies) and LAMP (1291 flies plus 138 nulliparous flies) analyses.ResultsThe LAMP assay successfully detected parasites in Chrysops fed on volunteers with both low and high microfilariaemic loads. Field validation and surveillance studies revealed LAMP-based infection rates ranging from 0.5 to 31.6%, with the lowest levels in SW 2 and the highest infection rates in SW 1. The LAMP assay detected significantly higher infection rates than microscopy in four of the five study sites.ConclusionThis study demonstrated the potential of LAMP as a simple surveillance tool. It was found to be more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

Project description:BACKGROUND:Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. METHODS:In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. RESULTS:Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (? = 0.490), both IMDM (? = 0.256) and DMEM (? = 0.198) media and the protein supplements NCS (? = 0.052) and FBS (? = 0.022); while for L. loa L3, in addition to feeder cells (? = 0.259) and both IMDM (? = 0.401) and DMEM (? = 0.385) media, the protein supplements BSA (? = 0.029) were found important in maintaining the worm motility. CONCLUSIONS:The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

Project description:BackgroundLoiasis is a filarial disease caused Loa loa. The main vectors are Chrysops silacea and C. dimidiata which are confined to the tropical rainforests of Central and West Africa. Loiasis is a mild disease, but individuals with high microfilaria loads may suffer from severe adverse events if treated with ivermectin during mass drug administration campaigns for the elimination of lymphatic filariasis and onchocerciasis. This poses significant challenges for elimination programmes and alternative interventions are required in L. loa co-endemic areas. The control of Chrysops has not been considered as a viable cost-effective intervention; we reviewed the current knowledge of Chrysops vectors to assess the potential for control as well as identified areas for future research.ResultsWe identified 89 primary published documents on the two main L. loa vectors C. silacea and C dimidiata. These were collated into a database summarising the publication, field and laboratory procedures, species distributions, ecology, habitats and methods of vector control. The majority of articles were from the 1950-1960s. Field studies conducted in Cameroon, Democratic Republic of Congo, Equatorial Guinea, Nigeria and Sudan highlighted that C. silacea is the most important and widespread vector. This species breeds in muddy streams or swampy areas of forests or plantations, descends from forest canopies to feed on humans during the day, is more readily adapted to human dwellings and attracted to wood fires. Main vector targeted measures proposed to impact on L. loa transmission included personal repellents, household screening, indoor residual spraying, community-based environmental management, adulticiding and larviciding.ConclusionsThis is the first comprehensive review of the major L. loa vectors for several decades. It highlights key vector transmission characteristics that may be targeted for vector control providing insights into the potential for integrated vector management, with multiple diseases being targeted simultaneously, with shared human and financial resources and multiple impact. Integrated vector management programmes for filarial infections, especially in low transmission areas of onchocerciasis, require innovative approaches and alternative strategies if the elimination targets established by the World Health Organization are to be achieved.

Project description:BACKGROUND:Loiasis is caused by the filarial parasite Loa loa, which is widespread through Central and West Africa and largely confined the tropical equatorial rainforests. The tabanid flies Chrysops silacea and Chrysops dimidiata are the main vectors driving transmission. This study aimed to better define the spatial distribution and ecological niche of the two vectors to help define spatial-temporal risk and target appropriate, timely intervention strategies for filariasis control and elimination programmes. METHODS:Chrysops spp. distributions were determined by collating information from the published literature into a database, detailing the year, country, locality, latitude/longitude and species collected. Environmental factors including climate, elevation and tree canopy characteristics were summarised for each vector from data obtained from satellite modelled data or imagery, which were also used to identify areas with overt landcover changes. The presence of each Chrysops vector was predicted using a maximum entropy species distribution modelling (MaxEnt) method. RESULTS:A total of 313 location-specific data points from 59 published articles were identified across seven loiasis endemic countries. Of these, 186 sites were included in the climate and elevation analysis, and due to overt landcover changes, 83 sites included in tree canopy analysis and MaxEnt model. Overall, C. silacea and C. dimidiata were found to have similar ranges; annual mean temperature (24.6 °C and 24.1 °C, respectively), annual precipitation (1848.6 mm and 1868.8 mm), elevation (368.8 m and 400.6 m), tree canopy cover (61.4% and 66.9%) and tree canopy height (22.4 m and 25.1 m). MaxEnt models found tree canopy coverage was a significant environmental variable for both vectors. CONCLUSIONS:The Chrysops spp. database and large-scale environmental analysis provides insights into the spatial and ecological parameters of the L. loa vectors driving transmission. These may be used to further delineate loiasis risk, which will be important for implementing filariasis control and elimination programmes in the equatorial rainforest region of Central and West Africa.

Dataset Information

Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea.

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Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea.

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