Effects of host vimentin on Eimeria tenella sporozoite invasion

Background Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa, which has caused great economic loss to the poultry breeding industry. Host vimentin is a key protein in the process of infection of many pathogens. In an earlier phosphorylation proteomics study, we found that the phosphorylation level of host vimentin was significantly regulated after Eimeria tenella sporozoite infection. Therefore, we explored the role of host vimentin in the invasion of host cells by sporozoites. Methods Chicken vimentin protein was cloned and expressed. We used qPCR, western blotting, and indirect immunofluorescence to detect levels of mRNA transcription, translation, and phosphorylation, and changes in the distribution of vimentin after E. tenella sporozoite infection. The sporozoite invasion rate in DF-1 cells treated with vimentin polyclonal antibody or with small interfering RNA (siRNA), which downregulated vimentin expression, was assessed by an in vitro invasion test. Results The results showed that vimentin transcription and translation levels increased continually at 6–72 h after E. tenella sporozoite infection, and the total phosphorylation levels of vimentin also changed. About 24 h after sporozoite infection, vimentin accumulated around sporozoites in DF-1 cells. Treating DF-1 cells with vimentin polyclonal antibody or downregulating vimentin expression by siRNA significantly improved the invasion efficiency of sporozoites. Conclusion In this study, we showed that vimentin played an inhibitory role during the invasion of sporozoites. These data provided a foundation for clarifying the relationship between Eimeria and the host. Graphical Abstract

Assessing Anopheles vector species diversity and transmission of malaria in four health districts along the borders of Côte d’Ivoire

Background Although malaria and Anopheles mosquito vectors are highly prevalent in Côte d’Ivoire, limited data are available to help understand the malaria vector density and transmission dynamics in areas bordering the country. To address this gap, the Anopheles mosquito species diversity, the members of the Anopheles gambiae complex and the transmission of malaria were assessed in four health districts along the borders of Côte d’Ivoire. Methods From July 2016 through December 2016 and July 2017 through December 2017, adult Anopheles mosquitoes were collected in four health districts of Côte d’Ivoire (Aboisso, Bloléquin, Odienné and Ouangolodougou) using standardized window exit trap (WET) and pyrethrum knockdown spray collection (PSC) methods. The collected mosquitoes were identified morphologically at species level and the members of the An. gambiae complex were separated using short interspersed nuclear element-based polymerase chain reaction (SINE-PCR). Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l. and Anopheles nili specimens were analysed for malaria Plasmodium parasite detection using the cytochrome oxidase I gene (COX-I), and malaria prevalence among human population through local Ministry of Health (MoH) statistical yearbooks. Results A total of 281 female Anopheles were collected in Aboisso, 754 in Bloléquin, 1319 in Odienné and 2443 in Ouangolodougou. Seven Anopheles species were recorded including An. gambiae s.l. (94.8–99.1%) as the main vector, followed by An. funestus s.l. (0.4–4.3%) and An. nili (0–0.7%). Among An. gambiae s.l., Anopheles coluzzii represented the predominant species in Aboisso (89.2%) and Bloléquin (92.2%), while An. gambiae sensu stricto (s.s.) was the major species in Odienné (96.0%) and Ouangolodougou (94.2%). The Plasmodium sporozoite infection rate in An. gambiae s.l. was highest in Odienné (11.0%; n = 100) followed by Bloléquin (7.8%, n = 115), Aboisso (3.1%; n = 65) and Ouangologoudou (2.5%; n = 120). In An. funestus s.l., Plasmodium falciparum sporozoite infection rate was estimated at 6.2% (n = 32) in Bloléquin, 8.7% (n = 23) in Odienné. No An. funestus s.l. specimens were found infected with P. falciparum sporozoite infection in Ouangolodougou and Aboisso. No P. falciparum sporozoite was detected in An. nili specimens in the four health districts. Among the local human populations, malaria incidence was higher in Odienné (39.7%; n = 45,376) and Bloléquin (37.6%; n = 150,205) compared to that in Ouangolodougou (18.3%; n = 131,629) and Aboisso (19.7%; n = 364,585). Conclusion Anopheles vector species diversity, abundance and Plasmodium sporozoite infection were high within the health districts along the borders of the country of Côte d’Ivoire, resulting in high malaria transmission among the local populations. Anopheles gambiae s.l. and An. funestus s.l. were found to be highly infected with Plasmodium in the health districts of Bloléquin and Odienné where higher malaria incidence was observed than the other districts. This study provides important information that can be used to guide Côte d’Ivoire National Malaria Control Programme for vector control decision-making, mainly in districts that are at the country borders.

Anti-TRAP/SSP2 monoclonal antibodies can inhibit sporozoite infection and enhance protection of anti-CSP monoclonal antibodies

Vaccine-induced sterilizing protection from infection with the Plasmodium parasite, the pathogen that causes malaria, will be an essential tool in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection. While such disease reduction is important, these results also indicate that further improvements to vaccines based solely on CSP will likely yield diminishing benefits towards the goal of durable, infection-blocking immunity. Here, we show that monoclonal antibodies (mAbs) recognizing the sporozoite protein TRAP/SSP2 across the major protein domains exhibit a range of inhibitory capacity and that these mAbs can augment CSP-based protection despite delivering no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.

Larval ecology and bionomics of Anopheles funestus in highland and lowland sites in western Kenya

Background An. funestus is a major Afrotropical vector of human malaria. This study sought to investigate the larval ecology, sporozoite infection rates and blood meal sources of An. funestus in western Kenya. Methods Larval surveys were carried out in Bungoma (Highland) and Kombewa (lowland) of western Kenya. Aquatic habitats were identified, characterized, georeferenced and carefully examined for mosquito larvae and predators. Indoor resting mosquitoes were sampled using pyrethrum spray catches. Adults and larvae were morphologically and molecularly identified to species. Sporozoite infections and blood meal sources were detected using real-time PCR and ELISA respectively. Results Of the 151 aquatic habitats assessed, 62/80 (78%) in Bungoma and 58/71(82%) in Kombewa were positive for mosquito larvae. Of the 3,193 larvae sampled, An. funestus larvae constitute 38% (1224/3193). Bungoma recorded a higher number of An. funestus larvae (85%, 95%, CI, 8.722–17.15) than Kombewa (15%, 95%, CI, 1.33–3.91). Molecular identification of larvae showed that 89% (n = 80) were An. funestus. Approximately 59%, 35% and 5% of An. funestus larvae co-existed with An. gambiae s.l, Culex spp and An. coustani in the same habitats respectively. Of 1,221 An. funestus s.l adults sampled, molecular identifications revealed that An. funestus constituted 87% (n = 201) and 88% (n = 179) in Bungoma and Kombewa, respectively. The Plasmodium falciparum sporozoite rate of An. funestus in Bungoma and Kombewa was 2% (3/174) and 1% (2/157), respectively, and the human blood index of An. funestus was 84% (48/57) and 89% (39/44) and for Bungoma and Kombewa, respectively. Conclusion Man-made ponds had the highest abundance of An. funestus larvae. Multiple regression and principal component analyses identified the distance to the nearest house as the key environmental factor associated with the abundance of An. funestus larvae in aquatic habitats. This study serves as a guide for the control of An. funestus and other mosquito species to complement existing vector control strategies.

Magnitude, specificity and avidity of sporozoite specific antibodies associate with protection status and distinguish among RTS,S/AS01 dose regimens

Background The malaria vaccine, RTS,S/AS01, demonstrated an enhanced efficacy (86.7%) in a delayed third fractional dose (0.1.7Fx) regimen in controlled human malaria infection (CHMI) trials compared to a standard full dose (0.1.2) regimen (62.5%). In order to understand the humoral component of the RTS,S/AS01 vaccine-induced protection against sporozoite infection in these two regimens, we investigated the serum antibody dynamics of 0.1.2 and 0.1.7Fx groups vaccinees. Methods The specific binding responses (magnitude) and dissociation rates (avidity) of serum antibodies interaction with a recombinant Plasmodium falciparum circumsporozoite protein (CSP) and peptides corresponding to the central repeat region (NANP6), the C-terminal region (PF16) and the N-terminal junction (N-interface) of CSP, respectively, were measured using a Biolayer Interferometry (BLI) assay. Results On the day of challenge, higher NANP6 specific antibody responses were associated with protection in the 0.1.2 group. Contrarily, slower antibody dissociation rates for CSP and PF16 binding were observed in the protected 0.1.7Fx group. Protected vaccinees of both groups exhibited 2 to 3-fold higher N-interface peptide binding antibody responses. Conclusions Unlike the standard dose, the delayed-fractional third dose of RTS,S/AS01 induced higher avidity CSP and PF16 binding antibodies that were associated with protection against sporozoite infection. Clinical Trials registration NCT01857869

STK35L1 regulates Plasmodium infection and expression of cell cycle genes during liver stage of malaria

Protein kinases of both the parasite and host are crucial in parasite invasion and survival and might be drug targets against drug-resistant malaria. STK35L1 was among the top five hits in kinome-wide screening, suggesting a role in malaria’s liver stage. The function of STK35L1 in malaria is not known yet. We found that STK35L1 was highly upregulated during the infection of P. berghei in HepG2 cells and mice liver. Knockdown of STK35L1 remarkably suppressed the sporozoite infection in hepatocytes. STAT3 is upregulated and phosphorylated during P. berghei sporozoites infection. We found that STAT3 activation is required for both STK35L1 and STAT3 upregulation. Furthermore, ten cell cycle genes were upregulated in the sporozoite-infected hepatocytes. Knockdown of STK35L1 completely inhibited the upregulation of these genes. We identified STK35L1 as a host kinase that plays an obligatory role in malaria’s liver stage. It may be a potential drug target against drug-resistant malaria.

Assessing Anopheles Vector Species Diversity and Transmission of Malaria in Cross-Border Areas of Côte d'Ivoire

Background Although malaria and Anopheles mosquito vectors are highly prevalent in Côte d'Ivoire, data are still lacking on disease transmission dynamics in cross-border areas. To address this lack of information, we assessed the Anopheles mosquito vector species diversity, the An. gambiae complex members and the transmission of malaria in four cross-border areas of Côte d'Ivoire.Method From July 2016 to December 2017, we collected adult Anopheles mosquitoes in four cross-border health districts of Côte d’Ivoire (Aboisso, Bloléquin, Odienné and Ouangolodougou) using standardized window exit trap (WET) and pyrethrum knockdown spray collection (PSC) methods. We identified collected mosquitoes morphologically at species level and An. gambiae complex members using short interspersed nuclear element-based polymerase chain reaction (SINE-PCR). We analyzed An. gambiae, An. funestus and An. nili specimens for malaria Plasmodium parasite infection using the cytochrome oxidase I gene (COX-I), and malaria prevalence among human population through local Ministry of Health (MoH) statistical yearbooks.Results In total, 281, 754, 1,319 and 2,443 specimens of Anopheles adult females were collected in Aboisso, Bloléquin, Odienné and Ouangolodougou, respectively. We found seven Anopheles species dominated by An. gambiae s.l. (94.8%-99.1%), followed by An. funestus (0.4%-4.3%) and An. nili (0%-0.7%). Among An. gambiae s.l., An. coluzzii predominated in Aboisso (89.2%) and Bloléquin (92.2%), while An. gambiae s.s. was present at the highest frequency in Odienné (96.0%) and Ouangolodougou (94.2%). The Plasmodium sporozoite infection rate in An. gambiae s.l. was highest in Odienné (11.0 %; n = 100) followed by Bloléquin (7.8%, n = 115), Aboisso (3.1%; n = 65) and Ouangologoudou (2.5%; n = 120). In An. funestus, P. falciparum sporozoite infection rate was estimated at 6.2% (n = 32) in Bloléquin, 8.7% (n = 23) in Odienné. No An. funestus specimens were found infected with P. falciparum sporozoite infection in Ouangolodougou and Aboisso. No P. falciparum sporozoite was detected in An. nili specimens in the four health districts. Among the local human populations, malaria prevalence rate was higher in Odienné (39.7%; n = 45,376) and Bloléquin (37.6%; n = 150,205) compared with that in Ouangolodougou (18.3%; n = 131,629) and Aboisso (19.7%; n = 364,585).Conclusion In cross-border health districts of Côte d’Ivoire, Anopheles vector species diversity and abundance and Plasmodium sporozoite infection were high, thus resulting in high transmission of malaria to local populations. An. gambiae and An. funestus were found to be highly Plasmodium-infected in the health districts of Bloléquin and Odienné where malaria prevalence among humans was particularly high. This study provides important information that can be used to guide national malaria control programme strategies in Côte d’Ivoire, mainly in cross-border settings.

Evaluation of Near Infrared Spectroscopy for Sporozoite Detection in Mosquitoes Infected With Wild-strain Parasites From Asymptomatic Gametocyte Carriers in Kilifi Kenya

Background: Screening for Plasmodium spp. sporozoite infection in mosquitoes is routinely done using ELISA (enzyme-linked immunosorbent assay). Near infrared spectroscopy (NIRS), a fast and non-destructive method, has recently been shown to distinguish, with 95% accuracy, between uninfected and sporozoite-infected mosquitoes using laboratory strains of Plasmodium falciparum (PfN54). The aim of this present study was to further investigate the reproducibility of NIRS to identify sporozoite infection in mosquitoes infected using field isolates of P. falciparum gametocytes from asymptomatic carriers. Methods: Healthy individuals (aged 5 years and above) were screened for gametocytaemia by thick-smear microscopy in an area of moderate transmission along the Coast of Kenya between May and September 2018. Asymptomatic gametocyte carriers were recruited for mosquito feeding assays, direct membrane feeding (DMFA) and direct skin feeding (DFA), using insectary-reared Anopheles gambiae s.s (Kilifi strain). Mosquitoes were kept for 14-days post feeding after which they were scanned using NIRS and subsequently analysed for sporozoite infection using circumsporozoite-ELISA. Predictive models were explored using partial least square regressions (PLS).Results: Two hundred and ninety-nine (299) individuals were screened for malaria parasites, 74 (24.8%) were found with circulating asexual parasites, and 16 (5.4%) with P. falciparum gametocyte stages.Fourteen (14) asymptomatic gametocyte carriers were recruited to the study for mosquito feeding assays. A total of 134 (7%, 134/1881) sporozoite-infected mosquitoes were obtained from 9 successful experiments. Three different training datasets composed of infected and uninfected mosquitoes were analysed. The PLS models were unable to distinguish between sporozoite-infected and uninfected mosquitoes. A predictive model could not be generated.Conclusions: The results of this study were not consistent with previous published research on NIRS for detection of sporozoite infection in the same mosquito species and may reflect differences between laboratory and field conditions. The current findings indicate that methods for sporozoite detection should be tested on field isolates at an early stage in their development and are informative for future research seeking novel high-throughput methods for parasite detection in mosquitoes.

Evaluation of near infrared spectroscopy for sporozoite detection in mosquitoes infected with wild-strain parasites from asymptomatic gametocyte carriers in Kilifi Kenya

ABSTRACTBackgroundScreening for Plasmodium spp. sporozoite infection in mosquitoes is routinely done using ELISA (enzyme-linked immunosorbent assay). Near infrared spectroscopy (NIRS), a fast and non-destructive method, has recently been shown to distinguish, with 95% accuracy, between uninfected and sporozoite-infected mosquitoes using laboratory strains of Plasmodium falciparum (PfN54). The aim of this present study was to further investigate the reproducibility of NIRS to identify sporozoite infection in mosquitoes infected using field isolates of P. falciparum gametocytes from asymptomatic carriers.MethodsHealthy individuals (aged 5 years and above) were screened for gametocytaemia by thick-smear microscopy in an area of moderate transmission along the Coast of Kenya between May and September 2018. Asymptomatic gametocyte carriers were recruited for mosquito feeding assays, direct membrane feeding (DMFA) and direct skin feeding (DFA), using insectary-reared Anopheles gambiae s.s (Kilifi strain). Mosquitoes were kept for 14-days post feeding after which they were scanned using NIRS and subsequently analysed for sporozoite infection using circumsporozoite-ELISA. Predictive models were explored using partial least square regressions (PLS).ResultsTwo hundred and ninety-nine (299) individuals were screened for malaria parasites, 74 (24.8%) were found with circulating asexual parasites, and 16 (5.4%) with P. falciparum gametocyte stages. Fourteen (14) asymptomatic gametocyte carriers were recruited to the study for mosquito feeding assays. A total of 134 (7%, 134/1881) sporozoite-infected mosquitoes were obtained from 9 successful experiments. Three different training datasets composed of infected and uninfected mosquitoes were analysed. The PLS models were unable to distinguish between sporozoite-infected and uninfected mosquitoes. A predictive model could not be generated.ConclusionsThe results of this study were not consistent with previous published research on NIRS for detection of sporozoite infection in the same mosquito species and may reflect differences between laboratory and field conditions. The current findings indicate that methods for sporozoite detection should be tested on field isolates at an early stage in their development and are informative for future research seeking novel high-throughput methods for parasite detection in mosquitoes.

Anopheles gambiaeLackingAgTRIOInefficiently TransmitsPlasmodium bergheito Mice

ABSTRACTAntibodies to AgTRIO, a mosquito salivary protein, partially reduce the initialPlasmodiumburden in mice. We therefore silencedAgTRIOin mosquitoes and determined the relative contribution of AgTRIO to the ability ofAnopheles gambiaeto transmitPlasmodium bergheito mice. RNA interference-mediated silencing ofAgTRIO inA. gambiaeresulted in a 60% reduction inAgTRIOexpression. The decrease inAgTRIOexpression did not alter the burden ofPlasmodiumsporozoites in mosquito salivary glands. When experimentally injected into mice, sporozoites fromAgTRIO-silenced mosquitoes colonized the liver less effectively than sporozoites from control mosquitoes. Silencing ofAgTRIOdid not decrease the infectivity of sporozoitesin vitroor influence the expression of genes associated withPlasmodiumcell adhesion or traversal activity. AgTRIO decreased the expression of proinflammation cytokines by splenocytesin vitro. Moreover,in vivo, AgTRIO decreased the expression ofTNF-αwhen coinjected with sporozoites into the skin and there was moreTNF-αexpression at the bite site ofAgTRIOknockdown mosquitoes than at the bite site of control mosquitoes. AgTRIO therefore influences the local environment in the vertebrate host, which facilitatesPlasmodiumsporozoite infection in mice.