Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon.

BACKGROUND: Monitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors. METHODS: Anopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile. RESULTS: The study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.0%) and oocyst (14.4%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.9%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.4%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations. CONCLUSION: The emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

Adaptation of ELISA detection of Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in mosquitoes to a multiplex bead-based immunoassay.

BACKGROUND: Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites. METHODS: Recombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. RESULTS: Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8-4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined. CONCLUSIONS: The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Morphological and molecular characterization of Isospora amphiboluri (Apicomplexa: Eimeriidae), a coccidian parasite, in a central netted dragon (Ctenophorus nuchalis) (De Vis, 1884) in Australia.

An Isospora species, Isospora amphiboluri, originally described by Canon in 1967 and later by McAllister et al. (1995), was isolated from a central netted dragon (Ctenophorus nuchalis) housed at a wildlife rehabilitation centre in Perth, Western Australia. Sporulated oocysts of Isospora amphiboluri (n = 30) are spherical, 24.2 (26.5-23.0) µm in length and 23.9 (22.4-25.9) µm in width, with a shape index of 1.01. The bilayered oocyst wall is smooth and light-yellow in color. Polar granule, oocyst residuum and micropyle are absent. The sporocysts are lemon-shaped, 15.7 (15.2-18.0) × 10.2 (8.9-11.2) µm, with a shape index (length/width) of 1.53. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda half-moon-shaped. Each sporocyst contains four vermiform sporozoites arranged head to tail. The sporozoites are 11.7 (9.9-16.2) × 3.0 (2.4-3.5) µm, with a shape index (length/width) of 3.87. A sporocyst residuum is present. Sporozoites contain a central nucleus with a finely distributed granular residuum. Comparison of oocyst measurements and their features with other valid Isospora species from hosts in the Agamid family confirmed that this Isospora species is Isospora amphiboluri. Molecular characterization of I. amphiboluri at the 18S rRNA and MTCOI loci showed the highest similarity with I. amphiboluri from the central bearded dragon, 99.8% and 99.7% respectively. This is the first report of I. amphiboluri from a central netted dragon in Australia.

Indoor and outdoor malaria transmission in two ecological settings in rural Mali: implications for vector control.

BACKGROUND: Implementation and upscale of effective malaria vector control strategies necessitates understanding the multi-factorial aspects of transmission patterns. The primary aims of this study are to determine the vector composition, biting rates, trophic preference, and the overall importance of distinguishing outdoor versus indoor malaria transmission through a study at two communities in rural Mali. METHODS: Mosquito collection was carried out between July 2012 and June 2016 at two rural Mali communities (Dangassa and Koïla Bamanan) using pyrethrum spray-catch and human landing catch approaches at both indoor and outdoor locations. Species of Anopheles gambiae complex were identified by polymerase chain reaction (PCR). Enzyme-Linked -Immuno-Sorbent Assay (ELISA) were used to determine the origin of mosquito blood meals and presence of Plasmodium falciparum sporozoite infections. RESULTS: A total of 11,237 An. gambiae sensu lato (s.l.) were collected during the study period (5239 and 5998 from the Dangassa and Koïla Bamanan sites, respectively). Of the 679 identified by PCR in Dangassa, Anopheles coluzzii was the predominant species with 91.4% of the catch followed by An. gambiae (8.0%) and Anopheles arabiensis (0.6%). At the same time in Koïla Bamanan, of the 623 An. gambiae s.l., An. coluzzii accounted for 99% of the catch, An. arabiensis 0.8% and An. gambiae 0.2%. Human Blood Index (HBI) measures were significantly higher in Dangassa (79.4%; 95% Bayesian credible interval (BCI) [77.4, 81.4]) than in Koïla Bamanan (15.9%; 95% BCI [14.7, 17.1]). The human biting rates were higher during the second half of the night at both sites. In Dangassa, the sporozoite rate was comparable between outdoor and indoor mosquito collections. For outdoor collections, the sporozoite positive rate was 3.6% (95% BCI [2.1-4.3]) and indoor collections were 3.1% (95% BCI [2.4-5.0]). In Koïla Bamanan, the sporozoite rate was higher indoors at 4.3% (95% BCI [2.7-6.3]) compared with outdoors at 2.4% (95% BCI [1.1-4.2]). In Dangassa, corrected entomological inoculation rates (cEIRs) using HBI were 13.74 [95% BCI 9.21-19.14] infective bites/person/month (ib/p/m) at indoor, and 18.66 [95% BCI 12.55-25.81] ib/p/m at outdoor. For Koïla Bamanan, cEIRs were 1.57 [95% BCI 2.34-2.72] ib/p/m and 0.94 [95% BCI 0.43-1.64] ib/p/m for indoor and outdoor, respectively. EIRs were significantly higher at the Dangassa site than the Koïla Bamanan site. CONCLUSION: The findings in this work may indicate the occurrence of active, outdoor residual malaria transmission is comparable to indoor transmission in some geographic settings. The high outdoor transmission patterns observed here highlight the need for additional strategies to combat outdoor malaria transmission to complement traditional indoor preventive approaches such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) which typically focus on resting mosquitoes.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

Malaria vector species composition and entomological indices following indoor residual spraying in regions bordering Lake Victoria, Tanzania.

BACKGROUND: Vector control through long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) is a major component of the Tanzania national malaria control strategy. In mainland Tanzania, IRS has been conducted annually around Lake Victoria basin since 2007. Due to pyrethroid resistance in malaria vectors, use of pyrethroids for IRS was phased out and from 2014 to 2017 pirimiphos-methyl (Actellic® 300CS) was sprayed in regions of Kagera, Geita, Mwanza, and Mara. Entomological surveillance was conducted in 10 sprayed and 4 unsprayed sites to determine the impact of IRS on entomological indices related to malaria transmission risk. METHODS: WHO cone bioassays were conducted monthly on interior house walls to determine residual efficacy of pirimiphos-methyl CS. Indoor CDC light traps with or without bottle rotator were hung next to protected sleepers indoors and also set outdoors (unbaited) as a proxy measure for indoor and outdoor biting rate and time of biting. Prokopack aspirators were used indoors to capture resting malaria vectors. A sub-sample of Anopheles was tested by PCR to determine species identity and ELISA for sporozoite rate. RESULTS: Annual IRS with Actellic® 300CS from 2015 to 2017 was effective on sprayed walls for a mean of 7 months in cone bioassay. PCR of 2016 and 2017 samples showed vector populations were predominantly Anopheles arabiensis (58.1%, n = 4,403 IRS sites, 58%, n = 2,441 unsprayed sites). There was a greater proportion of Anopheles funestus sensu stricto in unsprayed sites (20.4%, n = 858) than in sprayed sites (7.9%, n = 595) and fewer Anopheles parensis (2%, n = 85 unsprayed, 7.8%, n = 591 sprayed). Biting peaks of Anopheles gambiae sensu lato (s.l.) followed periods of rainfall occurring between October and April, but were generally lower in sprayed sites than unsprayed. In most sprayed sites, An. gambiae s.l. indoor densities increased between January and February, i.e., 10-12 months after IRS. The predominant species An. arabiensis had a sporozoite rate in 2017 of 2.0% (95% CI 1.4-2.9) in unsprayed sites compared to 0.8% (95% CI 0.5-1.3) in sprayed sites (p = 0.003). Sporozoite rates were also lower for An. funestus collected in sprayed sites. CONCLUSION: This study contributes to the understanding of malaria vector species composition, behaviour and transmission risk following IRS around Lake Victoria and can be used to guide malaria vector control strategies in Tanzania.

A modified method for purification of Eimeria tenella sporozoites.

Coccidiosis is an economically important gastrointestinal disease in domestic fowl. Eimeria species are the causative agents of avian coccidiosis. Current challenges in management and prevention of eimeriosis enhance the need for research in this field. Sporozoite purification is a necessary step for Eimeria spp. in vitro infection models. Current alternatives such as DE-52 anion exchange chromatography and Percoll gradient require time and resources. We present a modified protocol consisting on vacuum filtration of sporozoites using a disposable 5-µL filter. Yield percentages were similar to those reported for Percoll gradient purification. By reducing time and efforts during sporozoite purification, it could be possible to increase resources in other areas of Eimeria studies.

Two- and Three-Dimensional Bioengineered Human Intestinal Tissue Models for Cryptosporidium.

Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions, support long-term growth, and complete the life cycle of the parasite. Previously, we developed a 3D silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2 weeks and resulted in completion of the life cycle of the parasite. However, transformed IECs are not representative of primary human IEC.Human intestinal enteroids (HIEs) are cultures derived from crypts that contain Lgr5+ stem cells isolated from human biopsies or surgical intestinal tissues; these established multicellular cultures can be induced to differentiate into enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and tuft cells. HIEs better represent human intestinal structure and function than immortalized IEC lines. Recently, significant progress has been made in the development of technologies to culture HIEs in vitro. When grown in a 3D matrix, HIEs provide a spatial organization resembling the native human intestinal epithelium. Additionally, they can be dissociated and grown as monolayers in tissue culture plates, permeable supports or silk scaffolds that enable mechanistic studies of pathogen infections. They can also be co-cultured with other human cells such as macrophages and myofibroblasts. The HIEs grown in these novel culture systems recapitulate the physiology, the 3D architecture, and functional diversity of native intestinal epithelium and provide a powerful and promising new tool to study Cryptosporidium-host cell interactions and screen for interventions ex vivo. In this chapter, we describe the 3D silk scaffold-based model using transformed IEC co-cultured with human intestinal myofibroblasts and 2D and 3D HIE-derived models of Cryptosporidium, also co-cultured with human intestinal myofibroblasts.

Coccidia of Columbiformes: a taxonomic review of its Eimeriidae species and Eimeria columbinae n. sp. from Columbina talpacoti (Temminck, 1809) from Brazil.

Coccidia (Chromista: Miozoa: Eimeriidae) of columbiform birds (Aves: Columbiformes) have been described since the end of the nineteenth century; however, some of these descriptions were poorly detailed or inconclusive. In this sense, the current work makes a detailed taxonomic revision reconsidering and organizing 18 Eimeria spp. and two Isospora spp. previously described or reported of Columbiformes. Along with this, a new species of Eimeria is morphologically and molecularly identified by the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and by the 18S small subunit ribosomal RNA (18S) gene from the ruddy ground-dove Columbina talpacoti (Temminck, 1809) in the Médio Paraíba region of the State of Rio de Janeiro, southeastern Brazil. Eimeria columbinae n. sp. has subspheroidal oocysts, 14.7 × 13.2 µm, with smooth, bi-layered wall, ~ 1.1 µm and length/width ratio of 1.1. Micropyle and oocyst residuum are present, but polar granule is absent. Sporocysts are ellipsoidal to slightly asymmetrical, 9.0 × 5.1 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum present and sporozoites with refractile body and nucleus. This is the 19th description of an eimerian from Columbiformes in the World, and the second to have a molecular identification of the COI and 18S genes.

Malaria surveillance from both ends: concurrent detection of Plasmodium falciparum in saliva and excreta harvested from Anopheles mosquitoes.

BACKGROUND: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). RESULTS: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. CONCLUSIONS: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.

Identification of a new vector species of avian haemoproteids, with a description of methodology for the determination of natural vectors of haemosporidian parasites.

BACKGROUND: Haemosporidian parasites are transmitted by dipteran blood-sucking insects but certain vectors remain unidentified for the great majority of described species. Sensitive PCR-based methods are often used for the detection of haemosporidian infection in wild-caught insects. However, this approach alone cannot distinguish between different sporogonic stages and thus is insufficient to demonstrate that the parasites produce the infective stage (sporozoite), which is essential for transmission. To prove that PCR-positive insects could act as vectors, the record of sporozoites is needed. We developed a methodology for the determination of natural vectors of avian Haemoproteus species and other haemosporidians. The essence of this approach is to apply PCR-based and microscopic diagnostic tools in parallel for sporozoite detection in insects. METHODS: Culicoides biting midges transmit avian Haemoproteus parasites, but certain insect species, which are involved in transmission, remain insufficiently investigated. Biting midges were collected in the wild and identified; parous females were dissected and preparations of thorax content containing salivary glands were prepared. Remnants of the dissected midges were screened using PCR-based methods. Only thorax preparations of PCR-positive biting midges were examined microscopically. RESULTS: In total, 460 parous females belonging to 15 species were collected and dissected. DNA of haemosporidians was detected in 32 (7%) of dissected insects belonging to 7 species. Of the thorax samples PCR-positive for Haemoproteus parasites, two preparations were microscopically positive for sporozoites. Both biting midges were Culicoides kibunensis. Haemoproteus pallidus (hPFC1) was identified, indicating that transmission of this infection occurs at the study site. It was proved that seven species of biting midges take bird blood meals naturally in the wild. CONCLUSIONS: Culicoides kibunensis is a new vector species of avian haemoproteids and is a natural vector of H. pallidus. Numerous studies have identified vectors of Haemoproteus parasites experimentally; however, this is the first direct identification of a natural vector of Haemoproteus infection in the Old World. We suggest using the described methodology for vector research of Haemoproteus and other haemosporidians in the wild.

Temporal escalation of Pyrethroid Resistance in the major malaria vector Anopheles coluzzii from Sahelo-Sudanian Region of northern Nigeria.

Despite the highest global burden of malaria, information on bionomics and insecticide resistance status of malaria vectors is grossly lacking in the densely populated Sahelo-Sudanian region of Nigeria. To support evidence-based vector control we characterised transmission and resistance profiles of Anopheles coluzzii populations from three sites in northern Nigeria. High sporozoite infection (~19.51%) was found in the An. coluzzii populations. A high pyrethroid resistance was observed with only 1% mortality against deltamethrin, a high LD50 (96.57 µg/ml), and a high LT50 (170.27 min, resistance ratio of ~51 compared with the fully susceptible Ngoussou colony). Moderate carbamate resistance was observed. Synergist bioassays significantly recovered deltamethrin susceptibility implicating CYP450s (mortality = 85%, χ2 = 134.04, p < 0.0001) and esterases (mortality = 56%, χ2 = 47.31, p < 0.0001). Reduced bed net efficacy was also observed, with mortalities on exposure to the roof of PermaNet3.0 (PBO + deltamethrin) more than 22 times compared to the side panel (deltamethrin). TaqMan genotyping revealed a high frequency of 1014F kdr mutation (82%) with significant difference in genotype distribution associated with permethrin resistance [OR = 4.69 (CI:1.53-14.35, χ2 = 8.22 p = 0.004]. Sequencing of exons 18-21 of the VGSC led to detection of two additional nonsynonymous mutations, Ile10148Asn and Ser1156Gly. These findings highlight the threats posed by the highly resistant An. coluzzii to malaria control in Nigeria.

Preliminary studies on in vitro methods for the evaluation of anticoccidial efficacy/resistance in ruminants.

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs.

Protozoan parasites in Culex pipiens mosquitoes in Vienna.

Avian malaria (Plasmodium spp.) and kinetoplastid (Trypanosoma spp.) parasites are common vector-borne pathogens in birds worldwide; however, knowledge about vector competence of different mosquito species is currently lacking. For a pilot project examining vector competence of mosquitoes of the Culex pipiens complex and Culex torrentium for protozoan parasites in the city of Vienna, 316 individual mosquitoes were sampled in the months June-August 2017 around the campus of the Veterinary University of Vienna. Since vector competence for avian Plasmodium can only be ascertained by finding infectious sporozoites in mosquito salivary glands, special emphasis was on examining these, or at least insect thoraxes, which contain the salivary glands. After species identification, the mosquitoes were processed in three different ways to determine the best method of visually detecting protozoan parasites in salivary glands: (1) microscopic examination of individual, fixed and Giemsa-stained salivary glands, (2) microscopic examination of stained sections of individually fixed and embedded mosquito thoraxes and (3) stained sections of individual whole insects. Material from all three groups was also subjected to PCR to detect avian haemosporidian and trypanosomatid parasite DNA. PCR was performed on all 316 collected mosquitoes, with 37 pools (n = 2-10) of 263 individuals and 53 single individuals in all together 90 PCR reactions. Avian Plasmodium was found in 18 (20%) and trypanosomatid parasites were found in 10 (11.1%) of the examined samples and pools yielded a higher proportion of positives than did individual samples. Six different species of protozoan parasites were identified, namely Plasmodium vaughani SYAT05 which was the most common, P. elongatum GRW6, P. relictum SGS1, Trypanosoma avium, T. culicavium and Crithidia dedva. Seventy-seven mosquito salivary glands were dissected and stained with Giemsa solution. Of these, one (1.3%) featured sporozoites and one (1.3%) trypanosomatid parasites. While the trypanosomes were identified as T. avium, the avian Plasmodium species were present in a mixed infection with P. vaughani SYAT05 as the dominant species. In conclusion, mosquitoes of the Culex pipiens complex are very likely vectors of different avian Plasmodium and Trypanosoma species and PCR was the most successful and reliable method for parasite detection in mosquito samples, delivering higher rates and more accurate results. The visual detection of parasite stages in the salivary glands was more difficult and only a few specimens were detected using Giemsa stain and chromogenic in situ hybridization. For further studies on vector competence of different protozoan parasites in mosquitoes, the use of PCR-based methods would be preferable.

Spatial-temporal heterogeneity in malaria receptivity is best estimated by vector biting rates in areas nearing elimination.

BACKGROUND: Decisions on when vector control can be withdrawn after malaria is eliminated depend on the receptivity or potential of an area to support vector populations. To guide malaria control and elimination programmes, the potential of biting rates, sporozoite rates, entomological inoculation rates and parity rates to estimate malaria receptivity and transmission were compared within and among geographically localised villages of active transmission in the Western Province of the Solomon Islands. RESULTS: Malaria transmission and transmission potential was heterogeneous in both time and space both among and within villages as defined by anopheline species composition and biting densities. Biting rates during the peak biting period (from 18:00 to 00:00 h) of the primary vector, Anopheles farauti, ranged from less than 0.3 bites per person per half night in low receptivity villages to 26 bites per person in highly receptive villages. Within villages, sites with high anopheline biting rates were significantly clustered. Sporozoite rates provided evidence for continued transmission of Plasmodium falciparum, P. vivax and P. ovale by An. farauti and for incriminating An. hinesorum, as a minor vector, but were unreliable as indicators of transmission intensity. CONCLUSIONS: In the low transmission area studied, sporozoite, entomological inoculation and parity rates could not be measured with the precision required to provide guidance to malaria programmes. Receptivity and potential transmission risk may be most reliably estimated by the vector biting rate. These results support the meaningful design of operational research programmes to ensure that resources are focused on providing information that can be utilised by malaria control programmes to best understand both transmission, transmission risk and receptivity across different areas.

Mind the weather: a report on inter-annual variations in entomological data within a rural community under insecticide-treated wall lining installation in Kwara State, Nigeria.

BACKGROUND: Entomological indices within a specific area vary with climatic factors such as rainfall, temperature and relative humidity. Contributions of such weather parameter fluctuations to the changes in entomological data obtained within a community under implementation of a promising vector control intervention should be taken into account. This study reports on inter-annual changes in entomological indices within two rural communities, one of which was under insecticide-treated durable wall lining (DL) installation. METHODS: Community-wide DL installation was followed by monthly meteorological data and pyrethrum spray mosquito collections for 2 years in intervention and a similar neighbouring community (control). Human blood meal and sporozoite ELISA tests were conducted on female mosquitoes collected alongside PCR identification of subsamples. Mosquitoes collected at the intervention site were tested in cone susceptibility assays against subsamples of installed DL materials collected on a 6-monthly basis for 2 years. Deltamethrin susceptibility of Anopheles mosquitoes from the intervention site was determined before and after DL installation. Entomological indices in the first and second years were compared within each site. RESULTS: Rainfall in the study area increased significantly (t = -3.45, df = 11, P = 0.005) from first to second year. Correlation between rainfall and Anopheles densities in both sites were significant (r = 0.681, P < 0.001). Mosquitoes collected at the intervention site were susceptible (100%) to deltamethrin at baseline but resistant (92%) in the second year. However, subsamples of installed DL materials remained effective (100% mortality) against Anopheles mosquitoes from the intervention site throughout the 6-monthly cone assay exposures. Monthly pyrethrum spray collections showed significant increase in Anopheles densities from first to second year in the control (6.36 ± 1.61 vs 7.83 ± 2.39; t = -3.47, df = 11, P = 0.005), but not in the intervention (2.83 ± 1.86 vs 4.23 ± 3.31; t = -2.03, df = 11, P = 0.067) community. However, mean annual mosquito man-biting rates increased significantly in both intervention (0.88 ± 0.18 vs 1.06 ± 0.38; F(1, 10) = 9.50, P = 0.012) and control (1.45 ± 0.31 vs 1.61 ± 0.34; F(1, 10) = 10.18, p = 0.010) sites along with increase (≥ 1.6 times) in sporozoite rates within intervention (0-2.13%) and control (2.56-4.04%) communities. CONCLUSIONS: The slight increase in vector density, induced by significant increase in rainfall, led to increased sporozoite infection and significantly increased man-biting rates within the intervention site. These reveal the need for incorporation of integrated vector management strategies to complement DL installation especially in regions with high rainfall and mosquito density. Promising vector control tools such as DL should be evaluated on a long-term basis to reveal the possible effect of weather parameters on control performance and also allow for holistic recommendations.

Detection of malaria sporozoites expelled during mosquito sugar feeding.

Malaria is a severe disease of global importance transmitted by mosquitoes of the genus Anopheles. The ability to rapidly detect the presence of infectious mosquitoes able to transmit malaria is of vital importance for surveillance, control and elimination efforts. Current methods principally rely on large-scale mosquito collections followed by labour-intensive salivary gland dissections or enzyme-linked immunosorbent (ELISA) methods to detect sporozoites. Using forced salivation, we demonstrate here that Anopheles mosquitoes infected with Plasmodium expel sporozoites during sugar feeding. Expelled sporozoites can be detected on two sugar-soaked substrates, cotton wool and Whatman FTA cards, and sporozoite DNA is detectable using real-time PCR. These results demonstrate a simple and rapid methodology for detecting the presence of infectious mosquitoes with sporozoites and highlight potential laboratory applications for investigating mosquito-malaria interactions. Our results indicate that FTA cards could be used as a simple, effective and economical tool in enhancing field surveillance activities for malaria.

Continuous Supply of Plasmodium vivax Sporozoites from Colonized Anopheles darlingi in the Peruvian Amazon.

In vitro culture of Plasmodium vivax liver stages underlies key understandings of the fundamental biology of this parasite, particularly the latent, hyponozoite stage, toward drug and vaccine development. Here, we report systematic production of Plasmodium vivax sporozoites in colonized Anopheles darlingi mosquitoes in the Peruvian Amazon. Human subject-derived P. vivax-infected blood was fed to Anopheles darlingi females using standard membrane feedings assays. Optimizing A. darlingi infection and sporozoite production included replacement of infected patient donor serum with naïve donor serum, comparing anticoagulants in processing blood samples, and addition of penicillin-streptomycin and ATP to infectious blood meals. Replacement of donor serum by naïve serum in the P. vivax donor blood increased oocysts in the mosquito midgut, and heparin, as anticoagulant, was associated with the highest sporozoite yields. Maintaining blood-fed mosquitoes on penicillin-streptomycin in sugar significantly extended mosquito survival which enabled greater sporozoite yield. In this study, we have shown that a robust P. vivax sporozoite production is feasible in a malaria-endemic setting where infected subjects and a stable A. darlingi colony are brought together, with optimized laboratory conditions.