Membrane Feeding Research Articles

Vector competence of Culex quinquefasciatus from Santiago Island, Cape Verde, to West Nile Virus: exploring the potential effect of the vector native Wolbachia

Abstract Background Culex quinquefasciatus plays a crucial role as a vector of West Nile virus (WNV). This mosquito species is widely distributed in Cape Verde, being found in all inhabited islands of the archipelago. However, no data are currently available on the susceptibility of the local mosquito population to WNV. This study aimed to assess the vector competence of Cx. quinquefasciatus mosquitoes from Santiago Island, Cape Verde, for WNV and to explore the potential impact of its native Wolbachia on virus transmission. Methods Wolbachia-infected and uninfected Cx. quinquefasciatus female mosquitoes were exposed to WNV lineage 1 PT6.39 strain using a Hemotek membrane feeding system. Mosquito samples, including the body, legs, wings and saliva, were collected at days 7, 14 and 21 post-infection (dpi) to assess WNV infection through one-step quantitative real-time PCR (RT-qPCR). Results Culex quinquefasciatus from Cape Verde exhibited high susceptibility to the tested strain of WNV. Also, treated females without their native Wolbachia exhibited significantly higher WNV load in their bodies and greater dissemination rate at 7 dpi than their wild-type counterparts carrying Wolbachia. Conclusions The high susceptibility to WNV of Cx. quinquefasciatus from Cape Verde poses a potential risk for virus transmission in the archipelago. However, Wolbachia infection in this mosquito species seems to confer protection against WNV dissemination in the early stages of viral infection. Additional research is required to uncover the mechanisms driving this protection and its potential impact on WNV transmission. Graphical abstract

Simplified Plasmodium falciparum membrane feeding assay using small Petri dishes and gel warmers

Studies on Plasmodium falciparum transmission require blood-feeding infectious gametocytes to mosquitoes using standard membrane-feeding assays (SMFAs). SMFAs are routinely performed using electric heating coils or glass membrane feeders connected to a circulatory water bath using tubing and clamps. Each of these approaches is expensive and requires a complex setup, hence restricting the number of assays that can be performed simultaneously. Furthermore, existing methods cannot be easily applied in low-resource field settings. This study presents a low-cost and simplified method for feeding mosquitoes with an infectious blood meal using 35 mm Petri dishes where temperature is maintained by using reusable gel warmers. The intensity and prevalence of infection in mosquitoes (Anopheles stephensi and Anopheles gambiae) fed via a Petri dish overlaid with gel warmers were comparable to mosquitoes fed using standard glass membrane feeders. The methodology described here can be easily applied in low-resource and field settings due to its low cost, ease of set up, and use of easily available supplies, such as Petri dishes, and reusable gel warmers. We believe a wide range of laboratories can easily adapt this method for P. falciparum transmission studies.

Mosquito populations originating from nonendemic areas have the potential to transmit recently emerging Japanese encephalitis virus genotype IV

Japanese encephalitis virus (JEV) genotype IV (GIV) is one of the least common and most neglected genotypes worldwide, having been identified only on a few Indonesian islands until it was recently found to be the cause of outbreaks that occurred in several Australian states in early 2022. Given the limited availability of information, the vector range for JEV GIV remains unknown; thus, understanding this range could prove invaluable for future prevention efforts in new areas. Herein, we experimentally exposed four mosquito colonies originated from various countries with no previous reports of GIV to JEV GIV strain 19CxBa-83-Cv, which was isolated from Culex vishnui Theobald collected in Bali in 2019. At 7 and 14 days post-JEV GIV exposure through a membrane feeding method, mosquito bodies, head-wings-legs, and saliva were harvested for infection, dissemination, and transmission efficiency analyses. The results showed robust transmission efficiencies of the virus by Culex tritaeniorhynchus Giles (∼74%) and Aedes albopictus Skuse (∼52%) from Japan, followed by Culex quinquefasciatus Say from Vietnam (∼35%) and Culex pipiens form molestus from Turkey (∼18%). Although significant differences were observed, we found that the four mosquito species could transmit JEV GIV. The efficiency of biological transmission of this restricted genotype by mosquitoes from various origins suggests that these mosquito species could support localized transmission if the genotype were introduced to their respective areas. This study emphasizes the importance of remaining vigilant and continuing arbovirus surveillance in all locations.

Artemether–lumefantrine–amodiaquine or artesunate–amodiaquine combined with single low-dose primaquine to reduce Plasmodium falciparum malaria transmission in Ouélessébougou, Mali: a five-arm, phase 2, single-blind, randomised controlled trial

Triple artemisinin-based combination therapies (TACTs) can delay the spread of antimalarial drug resistance. Artesunate-amodiaquine is widely used for uncomplicated Plasmodium falciparum malaria. We therefore aimed to determine the safety and efficacy of artemether-lumefantrine-amodiaquine and artesunate-amodiaquine with and without single low-dose primaquine for reducing gametocyte carriage and transmission to mosquitoes. We did a five-arm, single-blind, phase 2randomised controlled trial at the Ouélessébougou Clinical Research Unit of the Malaria Research and Training Centre of the University of Sciences, Techniques and Technologies of Bamako in Mali. Eligible participants were aged 10-50years, with asymptomatic Pfalciparum microscopy-detected gametocyte carriage. Eligible participants were randomly allocated (1:1:1:1:1) to receive either artemether-lumefantrine, artemether-lumefantrine-amodiaquine, artemether-lumefantrine-amodiaquine plus primaquine, artesunate-amodiaquine, or artesunate-amodiaquine plus primaquine. Treatment regimens were administered on days 0, 1, and 2; primaquine was given as a single dose on day 0. All staff except the trial pharmacist and participants were masked to the treatment allocation. The primary outcome was the median percentage change in mosquito infection rate between pretreatment and 2days after treatment initiation, assessed by direct membrane feeding assay. Data were analysed using a per-protocol analysis. This study is registered with ClinicalTrials.gov, NCT05550909. Between Oct 16, 2022, and Dec 28, 2022, a total of 1249individuals were screened; of whom, 100were enrolled and randomly assigned to one of the five treatment groups (20 per group). Before treatment, 61 (61%) of 100participants were infectious to mosquitoes, with a median of 7·3% (IQR 3·2 to 23·5) of mosquitoes becoming infected. Among infectious participants, the median percentage reduction in mosquito infection rate between pretreatment and 2days after treatment was 100% (IQR 100 to 100) in the artemether-lumefantrine (p=0·0018), artemether-lumefantrine-amodiaquine (p=0·0018), and artemether-lumefantrine-amodiaquine plus primaquine (p=0·0009) treatment groups. In the artesunate-amodiaquine group the median percentage reduction in mosquito infection rate was only 32% (IQR -10·9to 79·4; p=0·19), whereas a 100% median reduction was seen in the artesunate-amodiaquine plus primaquine group (IQR 100 to 100; p=0·0009). At day 2, two (10%) of 20participants in the artemether-lumefantrine group, two (11%) of 19in the artemether-lumefantrine-amodiaquine group, and 15 (75%) of 20in the artesunate-amodiaquine group infected any number of mosquitoes whereas no infected mosquitoes were observed at this timepoint in the groups with primaquine. 85 (85%) of 100participants had atotal of 262adverse events during follow-up; of which, 181 (69%) were categorised as mild and 81 (31%) asmoderate. No serious adverse events were reported. Our findings support the effectiveness ofartemether-lumefantrine alone or as part of TACT for preventing nearly all human-mosquito malaria parasitetransmission within 48h. By contrast, substantial transmission was observed following treatment with artesunate-amodiaquine. The addition of a single low dose of primaquine blocks transmission to mosquitoes rapidly regardless of schizonticide. Bill & Melinda Gates Foundation.

Anopheles jamesii Supports Plasmodium vivax Sporogony under Laboratory Conditions.

Anopheles jamesii, considered to be a non-malaria vector, is an abundant mosquito species found in coastal India and several countries in Asia. In Goa, western India, An. jamesii is the dominant anopheline species by numbers, and in recent epidemiological surveys, they have tested positive for Plasmodium carriage. To assess An. jamesii's capacity as a malaria vector, we carried out six controlled membrane feeding assays with the F1 generation of field-caught An. jamesii and Plasmodium vivax-infected patient blood samples. As a control, the established local vector, Anopheles stephensi, was fed in parallel to compare the oocyst and sporozoite loads. The average oocyst load of An. jamesii was 3.29, while for An. stephensi it was 10.23. Furthermore, An. jamesii's blood feeding rate (21.7% versus 85%), oocyst positivity rate (60% versus 90.6%), and sporozoite positivity (45.16% versus 83%) were significantly lower (P <0.05, unpaired t test) than those of An. stephensi, suggesting a recent adaptation to support Plasmodium sporogony.

Differential sensitivity and specificity of Aedes aegypti and Anopheles gambiae to adenine nucleotide phagostimulants—an all-or-none response?

BackgroundThe decision to imbibe a blood meal is predominantly dependent on the sensitivity and specificity of haematophagous arthropods to blood-derived adenine nucleotides, in particular adenosine triphosphate (ATP). Despite previous efforts to identify and characterise the specificity and sensitivity to ATP and other adenine nucleotides, as well as the role of other blood-derived phagostimulants across the Culicidae, comparisons across species remain difficult.MethodsThe feeding response of the yellow fever mosquito Aedes aegypti and the African malaria vector Anopheles gambiae to adenine nucleotides in the presence of a carbonate buffer was assessed using a membrane feeding assay. The proportion of mosquitoes engorged and the volume imbibed by all mosquitoes was scored visually and spectrophotometrically. In addition, the proportion of prediuresing An. gambiae, as well as the volume engorged and prediuresed, was examined.ResultsAedes aegypti was more sensitive to adenine nucleotides than An. gambiae, but both species maintained specificity to these phagostimulants, demonstrating a dose-dependent bimodal feeding pattern, thereby expanding our understanding of the all-or-none blood-feeding hypothesis. Feeding on the bicarbonate buffer by An. gambiae—but not that of Ae. aegypti—demonstrated a species-specific variation in how blood phagostimulants are encoded. Adenine nucleotides, with and without bovine serum albumin, were observed to dose-dependently regulate the proportion of An. gambiae prediuresing and the volumes prediuresed but not volumes engorged.ConclusionsTaken together, the results of this study expand our understanding of how mosquitoes differentially assess and respond to blood meal constituents, and provide a basis for further physiological and molecular studies.Graphical

Revisiting the paradigm of anhematophagy in male mosquitoes.

Female mosquitoes are reproductively obligate bloodfeeders which feed on vertebrate blood to obtain nutrients required for egg production (driving transmission of vector-borne pathogens in the process), and which rely on plant sugars for their non-reproductive energy requirements. Male mosquitoes, on the other hand, are thought to rely exclusively on plant sugars for their energetic needs; indeed, this dichotomy is one of the central tenets of medical entomology. Here, we show that male Culex tarsalis and Aedes aegypti mosquitoes will readily take blood from a membrane feeder when reared under dehydration conditions with no toxic effects. Mosquitoes with impaired humidity detection do not increase their bloodfeeding rates when dehydrated compared to wild-type controls. While conventionally reared males ignore a human host, dehydrated males are attracted to and attempt to probe, with some success, although they cannot access host capillaries. However, they will take blood from a vertebrate host wound. When fed a blood meal containing West Nile virus, male mosquitoes can become infected with and orally transmit the pathogen at rates and titers equivalent to females. Finally, vertebrate DNA, likely from blood, was detected in wild-caught specimens of male Culex quinquefasciatus mosquitoes from Texas. These data suggest that under some circumstances male mosquitoes may be able to probe and/or ingest blood and transmit pathogens to vertebrate hosts, and that their role in maintaining pathogen transmission cycles should be re-examined.

Genotype distribution and allele frequency of thioester-containing protein 1(Tep1) and its effect on development of Plasmodium oocyst in populations of Anopheles arabiensis in Ethiopia.

Thioester-containing protein 1 (TEP1) is a crucial component of mosquitoes' natural resistance to parasites. To effectively combat malaria, there is a need to better understand how TEP1 polymorphism affects phenotypic traits during infections. Therefore, the purpose of this study was to determine the Tep1 genotype frequency in malaria vector populations from south-western Ethiopia and investigate its effect on Plasmodium oocyst development in Anopheles arabiensis populations. Using standard dippers, Anopheles mosquito larvae were collected from aquatic habitats in Asendabo, Arjo Dedessa, and Gambella in 2019 and 2020. Collected larvae were reared to adults and identified morphologically. Female An. gambiae s.l. were allowed to feed on infected blood containing the same number of gametocytes obtained from P. falciparum and P. vivax gametocyte-positive individuals using indirect membrane feeding methods. Polymerase Chain Reaction (PCR) was used to identify An. gambiae s.l. sibling species. Three hundred thirty An. gambiae s.l. were genotyped using Restricted Fragment Length Polymorphism (RFLP) PCR and sub samples were sequenced to validate the TEP1 genotyping. Among the 330 samples genotyped, two TEP1 alleles, TEP1*S1 (82% frequency) and TEP1*R1 (18% frequency), were identified. Three equivalent genotypes, TEP1*S1/S1, TEP1*R1/R1, and TEP1*S1/R1, had mean frequencies of 65.15%, 2.12%, and 32.73%, respectively. The nucleotide diversity was ranging from 0.36554 to 0. 46751 while haplotype diversity ranged from 0.48871 to 0.63161, across all loci. All sample sites had positive Tajima's D and Fu's Fs values. There was a significant difference in the TEP1 allele frequency and genotype frequency among mosquito populations (p < 0.05), except populations of Anopheles arabiensis from Asendabo and Gambella (p > 0.05). In addition, mosquitoes with the TEP1 *RR genotype were susceptible and produced fewer Plasmodium oocysts than mosquitoes with the TEP1 *SR and TEP1 *SS genotypes. The alleles identified in populations of An. arabiensis were TEP1*R1 and TEP1*S1. There was no significant variation in TEP1*R1 allele frequency between the high and low transmission areas. Furthermore, An. arabiensis carrying the TEP1*R1 allele was susceptible to Plasmodium infection. Further studies on vector-parasite interactions, particularly on the TEP1 gene, are required for vector control techniques.

A human-serum-free medium can induce more infectious P. falciparum gametocytes than a conventional human-serum-containing medium

Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions.

Leucinostatins from fungal extracts block malaria transmission to mosquitoes

BackgroundMalaria is a mosquito-transmitted disease that kills more than half a million people annually. The lack of effective malaria vaccines and recently increasing malaria cases urge innovative approaches to prevent malaria. Previously, we reported that the extract from the soil-dwelling fungus Purpureocillium lilacinum, a common fungus from the soil, reduced Plasmodium falciparum oocysts in Anopheles gambiae midguts after mosquitoes contacted the treated surface before feeding.MethodsWe used liquid chromatography to fraction fungal crude extract and tract the active fraction using a contact-wise approach and standard membrane feeding assays. The purified small molecules were analyzed using precise mass spectrometry and tandem mass spectrometry.ResultsWe isolated four active small molecules from P. lilacinum and determined them as leucinostatin A, B, A2, and B2. Pre-exposure of mosquitoes via contact with very low-concentration leucinostatin A significantly reduced the number of oocysts. The half-maximal response or inhibition concentration (EC50) via pre-exposure was 0.7 mg/m2, similar to atovaquone but lower than other known antimalarials. The inhibitory effect of leucinostatin A against P. falciparum during intraerythrocytic development, gametogenesis, sporogonic development, and ookinete formation, with the exception of oocyst development, suggests that leucinostatins play a part during parasite invasion of new cells.ConclusionsLeucinostatins, secondary metabolites from P. lilacinum disrupt malaria development, particular transmission to mosquitoes by contact. The contact-wise malaria control as a nonconventional approach is highly needed in malaria-endemic areas.Graphical

Simple supplementation of serum-free medium produces gametocytes of Plasmodium falciparum that transmit to mosquitoes

BackgroundHuman serum is a major component of Plasmodium falciparum culture medium, and can be replaced with AlbuMAX™ II, a lipid-rich bovine serum albumin, for asexual cultures. However, gametocytes produced without serum are poorly infective to mosquitoes. Serum suffers from high cost, limited availability, and variability in quality.MethodsSeveral commercially-available media supplements were tested for their ability to support parasite growth and production of P. falciparum (3D7) gametocytes in standard RPMI1640 medium containing 0.5% AlbuMAX. The impact on asexual growth and gametocyte production with each supplement was assessed and compared to standard RPMI1640 medium containing 10% human serum, as well as to medium containing 0.5% AlbuMAX alone. The infectivity of gametocytes produced with one supplement to Anopheles gambiae sensu stricto was assessed by standard membrane feeding assay and measuring both prevalence of infection and oocyst intensity.ResultsSupplementation of medium containing 0.5% AlbuMAX with five supplements did not affect asexual growth of P. falciparum, and four of the five supplements supported early gametocyte production. The supplement producing the highest number of gametocytes, ITS-X, was further investigated and was found to support the production of mature gametocytes. Infection prevalence and oocyst intensity did not differ significantly between mosquitoes given a membrane feed containing gametocytes grown in medium with 0.5% AlbuMAX + ITS-X and those grown in medium with 10% human serum. Infection prevalence and oocyst intensity was significantly higher in case of ITS–X supplementation when compared to AlbuMAX alone. Infectious gametocytes were also produced from two field clones using ITS–X supplementation.ConclusionsSerum-free medium supplemented with ITS-X was able to support the growth of gametocytes of P. falciparum that were as infectious to An. gambiae as those grown in medium with 10% serum. This is the first fully serum-free culture system able to produce highly infectious gametocytes, thereby removing the requirement for access to serum for transmission assays.

Anti-infectivity efficacy and pharmacokinetics of WHO recommended single low-dose primaquine in children with acute Plasmodium falciparum in Burkina Faso: study protocol

BackgroundPrimaquine (PQ) has activity against mature P. falciparum gametocytes and proven transmission blocking efficacy (TBE) between humans and mosquitoes. WHO formerly recommended a single transmission blocking dose of 0.75 mg/kg but this was little used. Then in 2012, faced with the emergence of artemisinin-resistant P. falciparum (ARPf) in SE Asia, the WHO recommended a lower dose of 0.25 mg/kg to be added to artemisinin-based combination therapy in falciparum-infected patients in low transmission areas. This dose was considered safe in glucose-6-phosphate dehydrogenase deficiency (G6PDd) and not requiring G6PD testing. Subsequent single low-dose primaquine (SLDPQ) studies have demonstrated safety in different G6PD variants. Dosing remains challenging in children under the age of 5 because of the paucity of PQ pharmacokinetic (PK) data. We plan to assess the anti-infectivity efficacy of SLDPQ using an allometrically scaled, weight-based regimen, with a target dose of 0.25 mg/kg, in children with acute uncomplicated falciparum malaria.MethodsThis study is an open label, randomised 1:1, phase IIb study to assess TBE, tolerability, pharmacokinetics and acceptability of artesunate pyronaridine (ASPYR) administered alone or combined with SLDPQ in 56 Burkinabe children aged ≥ 6 months– < 5 years, with uncomplicated P. falciparum and a haemoglobin (Hb) concentration of ≥ 5 g/dL. We will assess TBE, using direct membrane feeding assays (DMFA), and further investigate PQ pharmacokinetics, adverse events, Hb dynamics, G6PD, sickle cells, thalassaemia and cytochrome 2D6 (CYP2D6) status, acceptability of flavoured PQ [CAST—ClinSearch Acceptability Score Test®], and the population’s knowledge, attitude and practices on malaria.Expected results and discussionWe expect children to accept tablets, confirm the TBE and gametocytocidal effects of SLDPQ and then construct a PK infectivity model (including age, sex, baseline Hb, G6PD and CYP2D6 status) to define the dose response TBE relationship that may lead to fine tuning our SLDPQ regimen. Our study will complement others that have examined factors associated with Hb dynamics and PQ PK. It will provide much needed, high-quality evidence of SLDPQ in sick African children and provide reassurance that SLDPQ should be used as a strategy against emerging ARPf in Africa.Trial registrationISRCTN16297951. Registered on September 26, 2021

H2 and CO purification by CO2 removal from syngas coupling membrane module and water-absorption unit: An economic analysis

Hydrogen represents today a necessary alternative to fossil fuels to limit the greenhouse gas effect due to the CO2 emissions. In this work, a plant configuration able to get pure H2 and concentrated CO (about 94%) from a syngas mixture is proposed and simulated by coupling a selective membrane unit and a water absorption column. In addition, an economic assessment on the simulated plant is carried out to estimate the feasibility of this process in terms of economic potential, studying its dependence on several parameters, such as raw material price, membrane cost, membrane feed pressure, column temperature and feed flow rate.Separation performance is improved by the higher feed membrane pressure (greater H2 recovery and CO purity), whereas the increment of the column temperature especially affects the CO recovery. Hence, the economic potential presents a maximum with pressure and temperature (e.g., about 246ꞏ103 $/y at 40 bar of feed membrane pressure and about 282ꞏ103 $/y at 50 °C of column temperature), due to the balance between the higher compression/solvent cost and the increment of H2 or CO recovery. In addition, the simulated plant shows a positive economic potential in a wide range of syngas price (e.g., from 0.126 to 0.25 $/Nm3), being also not affected significantly by the cost of the palladium-based membrane module. A bigger plant size (i.e., feed flow rate from 10 to 100 kmol/h) can lead to a twelve times greater economic potential (from about 242ꞏ103 to about 2954ꞏ103 $/y at 30 bar), reducing the H2 purification cost and making the net present value positive.

Immunogenicity and transmission-blocking potential of quiescin sulfhydryl oxidase in Plasmodium vivax.

Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.

Cd loop fusion enhances the immunogenicity and the potential transmission blocking activity of Plasmodium falciparum generative cell specific 1 (GCS1) antigen

TBVs are suggested to inhibit parasite transmission from humans to Anopheles mosquitoes. For the transmission of Plasmodium parasite, a variety of factors are included in gametes fusion phase. In this step, conserved male-specific generative cell specific 1 antigen is necessary for fusion of cytoplasmic membranes of micro- and macro-gametocytes and zygot formation. The partial blocking activities of elicited antibodies against either the HAP2-GCS1 domain or the cd loop of this antigen have been recorded to hinder the transmission of Plasmodium species in Anopheles mid-gut. Thus, the objective of the present study was to investigate if the cd loop-fusion can enhance the quantity and quality of humoral and cellular immune responses against Plasmodium falciparum GCS1 in comparison to non-fusion antigen (without cd loop), in the adjuvanted and non-adjuvanted mouse groups. The immunogenicity of two constructs of P. falciparum generative cell specific 1 antigen, a fusion protein composed of cd loop and HAP2-GCS1 domain (cd-HAP) and another recombinant PfGCS1 containing solo HAP2-GCS1 domain (HAP2) were assessed to impede Plasmodium gametocytes integration before zygote formation. The antibodies profiling, titer, and avidity of induced antibodies were measured by the immunized mice sera, and the released cytokines (IL-5, TNF, and INF-γ) were analyzed in the supernatants of stimulated splenocytes. Furthermore, the inhibitory potency of the elicited antibodies against HAP2 and cd-HAP was measured during oocyst development by Standard Membrane Feeding Assay (SMFA). The comparative results in the present study showed the higher titer of IgG antibodies and IgG2a subclass, avidity, and transmission-reducing activity (TRA = 72.5 %) when mice were immunized by cd-HAP rather than HAP2. Moreover, our findings confirmed intensified Th1-directed immune responses in group 4 received cd-HAP/Poly(I:C). These findings declared the potential ability of cd loop fusion (cd-HAP) to upsurge humoral and cellular immune responses. However, the immune responses may switch to stronger Th1-type using alternative formulations. Explicitly, the cd-HAP-based vaccine may enhance the overall efficiency of immune responses and present a promising implementation in aiming malaria transmission.

Laboratory Management of Mammalian Hosts for Ixodes scapularis -Host-Pathogen Interaction Studies.

Due to their hematophagous life cycle, hard-bodied ticks including the genus Ixodes are a potential vector for numerous pathogenic organisms including bacteria, protozoa, viruses, and infectious prions. The natural geographic range of several hard tick species, includig Ixodes scapularis, has expanded over recent decades. Consequently, there is an ongoing need to maintain, feed, and propagate ticks for host-pathogen interaction studies to better understand and mitigate their impact on human and animal health. Artificial membrane feeding of hard ticks has advanced in recent years, has study design advantages, and should be used, when possible, to reduce animal use, but it also has several limitations that require the continued use of mammalian hosts including mice, guinea pigs, and rabbits. In this overview, we discuss the best management practices for these relevant species with respect to biosafety, health, and optimal host comfort when used in studies that depend on tick feeding. The capsule-jacket method is preferred over the ear sock-E-collar method of tick feeding on rabbit hosts because of better host health, comfort, and increased study versatility.

Evaluation of combination vaccines targeting transmission of Plasmodium falciparum and P. vivax

Transmission-blocking vaccines interrupting malaria transmission within mosquitoes represent an ideal public health tool to eliminate malaria at the population level. Plasmodium falciparum and P. vivax account for more than 90% of the global malaria burden, co-endemic in many regions of the world. P25 and P48/45 are two leading candidates for both species and have shown promising transmission-blocking activity in preclinical and clinical studies. However, neither of these target antigens as individual vaccines has induced complete transmission inhibition in mosquitoes. In this study, we assessed immunogenicity of combination vaccines based on P25 and P48/45 using a DNA vaccine platform to broaden vaccine specificity against P. falciparum and P. vivax. Individual DNA vaccines encoding Pvs25, Pfs25, Pvs48/45 and Pfs48/45, as well as various combinations including (Pvs25 + Pvs48/45), (Pfs25 + Pfs48/45), (Pvs25 + Pfs25), and (Pvs48/45 + Pfs48/45), were evaluated in mice using in vivo electroporation. Potent antibody responses were induced in mice immunized with individual and combination DNA vaccines, and specific antibody responses were not compromised when combinations of DNA vaccines were evaluated against individual DNA vaccines. The anti-Pvs25 IgG from individual and combination groups revealed concentration-dependent transmission-reducing activity (TRA) in direct membrane feeding assays (DMFA) using blood from P. vivax-infected donors in Brazil and independently in ex vivo MFA using Pvs25-transgenic P. berghei. Similarly, anti-Pfs25 and anti-Pfs48/45 IgGs from mice immunized with Pfs25 and Pfs48/45 DNA vaccines individually and in various combinations revealed antibody dose-dependent TRA in standard membrane feeding assays (SMFA) using culture-derived P. falciparum gametocytes. However, antibodies induced by immunization with Pvs48/45 DNA vaccines were ineffective in DMFA and require further vaccine construct optimization, considering the possibility of induction of both transmission-blocking and transmission-enhancing antibodies revealed by competition ELISA. These studies provide a rationale for combining multiple antigens to simultaneously target transmission of malaria caused by P. falciparum and P. vivax.

Malaria transmission blocking activity of Anopheles stephensi alanyl aminopeptidase N antigen formulated with MPL, CpG, and QS21 adjuvants.

Malaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model. The mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants. The results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

A comparative study on the vector competence of Anopheles stephensi from geographically distinct malarious and non-malarious urban areas in India to the malarial parasite, Plasmodium vivax.

Anopheles stephensi is responsible for the transmission of malaria in urban areas. Vector competence of An. stephensi from a non-malarious (Coimbatore) and highly malarious (Chennai) urban area were investigated to find out the reason for the non-transmission of malaria in Coimbatore. Vector competence (Susceptibility/refractoriness) of An. stephensi mosquitoes from Chennai (Malarious) and Coimbatore (Non-malarious), Tamil Nadu, India to Plasmodium vivax (Chennai) were investigated. Bioassays were carried out concurrently in both these strains by artificial membrane feeding technique using the same malaria infected blood. An. stephensi were dissected to observe infection in the midgut and salivary glands. The parasite infection, oocyst and sporozoite positivity rate, the oocyst load, correlation between male-female gametocyte ratio and infection, and Survival Analysis of parasitic stages during sporogony were analyzed and compared. The overall infection rate was 45.8 and 41.2 per cent in Chennai and Coimbatore. Oocyst count ranged from 1-80 and 1-208 respectively and not statistically significant. Oocyst positivity was high from Day 8-21in both strains. The Mean Survival Day (MSD) for oocyst was Day 14 in both strains. Sporozoite was observed in four experiments in each of the strains and the MSD for sporozoites was Day 20 and Day 17 in Chennai and Coimbatore. An. stephensi of Chennai and Coimbatore are equally susceptible to P. vivax infection and the non-transmission of malaria in Coimbatore can be attributed to external factors such as the presence of preferential breeding habitat, vector density, vector survival, and weather. The only difference observed was the comparatively shortened oocyst maturation time in the Coimbatore strain which requires further investigation.

High-flux ultrasonic processing for lithium separation using ionic liquid impregnated composite membranes

Battery industry, one of the most crucial components of the modern world, relies heavily on lithium production, and brines from the spent battery materials is one of the most important sources to exploit lithium. A new ultrasonic assisted membrane processing is proposed for lithium separation simulated brine. The effects of membrane composition, feed concentration, and ultrasonic conditions on the lithium extraction efficiency have been explored. The composite membrane including polysulfone (PSF) as the support and 1-alkyl-3-methylimidazolium hexafluorophosphate and tributyl phosphate as ionic liquid membrane. A porous PVC membrane has been used for prevention of the ILM loss. The optimal ultrasonic frequency is approximately 250 kHz, which matches the bulk modulus of the membrane and enhances the separation efficiency. Higher frequencies and optimized amplitude and pulse cycle settings further improve the lithium flux and selectivity. Moreover, higher flux and selectivity are achieved when separating lithium from alkali metal chlorides at higher feed concentrations, ranging from 250 ppm to 1000 ppm. The mechanism of enhanced lithium extraction by ultrasonics is attributed to the combination of microbubble formation, cavitation, and heat generation, which disrupt the concentration gradient and facilitate lithium transport across the membrane.