Parasite Multiplication Research Articles

Genomic survey maps differences in the molecular complement of vesicle formation machinery between Giardia intestinalis assemblages.

Giardia intestinalis is a globally important microbial pathogen with considerable public health, agricultural, and economic burden. Genome sequencing and comparative analyses have elucidated G. intestinalis to be a taxonomically diverse species consisting of at least eight different sub-types (assemblages A-H) that can infect a great variety of animal hosts, including humans. The best studied of these are assemblages A and B which have a broad host range and have zoonotic transmissibility towards humans where clinical Giardiasis can range from asymptomatic to diarrheal disease. Epidemiological surveys as well as previous molecular investigations have pointed towards critical genomic level differences within numerous molecular pathways and families of parasite virulence factors within assemblage A and B isolates. In this study, we explored the necessary machinery for the formation of vesicles and cargo transport in 89 Canadian isolates of assemblage A and B G. intestinalis. Considerable variability within the molecular complement of the endolysosomal ESCRT protein machinery, adaptor coat protein complexes, and ARF regulatory system have previously been reported. Here, we confirm inter-assemblage, but find no intra-assemblage variation within the trafficking systems examined. This variation includes losses of subunits belonging to the ESCRTIII as well as novel lineage specific duplications in components of the COPII machinery, ARF1, and ARFGEF families (BIG and CYTH). Since differences in disease manifestation between assemblages A and B have been controversially reported, our findings may well have clinical implications and even taxonomic, as the membrane trafficking system underpin parasite survival, pathogenesis, and propagation.

Progeny counter mechanism in malaria parasites is linked to extracellular resources.

Malaria is caused by the rapid proliferation of Plasmodium parasites in patients and disease severity correlates with the number of infected red blood cells in circulation. Parasite multiplication within red blood cells is called schizogony and occurs through an atypical multinucleated cell division mode. The mechanisms regulating the number of daughter cells produced by a single progenitor are poorly understood. We investigated underlying regulatory principles by quantifying nuclear multiplication dynamics in Plasmodium falciparum and knowlesi using super-resolution time-lapse microscopy. This confirmed that the number of daughter cells was consistent with a model in which a counter mechanism regulates multiplication yet incompatible with a timer mechanism. P. falciparum cell volume at the start of nuclear division correlated with the final number of daughter cells. As schizogony progressed, the nucleocytoplasmic volume ratio, which has been found to be constant in all eukaryotes characterized so far, increased significantly, possibly to accommodate the exponentially multiplying nuclei. Depleting nutrients by dilution of culture medium caused parasites to produce fewer merozoites and reduced proliferation but did not affect cell volume or total nuclear volume at the end of schizogony. Our findings suggest that the counter mechanism implicated in malaria parasite proliferation integrates extracellular resource status to modify progeny number during blood stage infection.

In Vitro and in Vivo Antimalarial Activity, Cytotoxicity and Phytochemical HRMS2 Profile of Plants from the Western Pará State, Brazilian Amazonia.

Ethnopharmacology and botanical taxonomy are valid criteria used to selecting plants for antimalarial bioprospection purposes. Based on these two criteria, ethanol extracts of 11 plants from Santarém City vicinities, Western Pará State, Brazilian Amazonia, had their in vitro antiplasmodial activity against chloroquine-resistant Plasmodium falciparum (W2 clone) assessed by the PfLDH method, whereas their cytotoxicity to HepG2-A16 cells was assessed through MTT assay. Acmella oleracea, Siparuna krukovii and Trema micrantha extracts disclosed the highest rate of parasite growth inhibition (90 %) in screening tests. In vivo antimalarial assays were conducted with these extracts against Plasmodium berghei (NK 65 strain) infected mice. Inhibition rate of parasite multiplication ranged from 41.4 % to 60.9 % at the lowest extract dose (25 mg/kg). HPLC-ESI-HRMS2 analyses allowed the putative identification of alkylamides, fatty acids, flavonoid glycosides and alkaloids in ethanol extracts deriving from these three plant species. Results pointed towards A. oleracea flowers ethanol extract as the most promising potential candidate to preclinical studies aiming the development of antimalarial phytomedicine.

Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa

Anopheles stephensi, an Asian malaria vector, continues to expand across Africa. The vector is now firmly established in urban settings in the Horn of Africa. Its presence in areas where malaria resurged suggested a possible role in causing malaria outbreaks. Here, using a prospective case–control design, we investigated the role of An. stephensi in transmission following a malaria outbreak in Dire Dawa, Ethiopia in April–July 2022. Screening contacts of patients with malaria and febrile controls revealed spatial clustering of Plasmodium falciparum infections around patients with malaria in strong association with the presence of An. stephensi in the household vicinity. Plasmodium sporozoites were detected in these mosquitoes. This outbreak involved clonal propagation of parasites with molecular signatures of artemisinin and diagnostic resistance. To our knowledge, this study provides the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa, highlighting the major public health threat posed by this fast-spreading mosquito.

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.

Toxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma, like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase PPKL appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

Development of a degree-day model to predict the growth of Anopheles stephensi (Diptera: Culicidae): implication for vector control management.

Anopheles stephensi is an efficient vector of malaria parasites in Iran. Despite its importance in malaria transmission, there is a scarcity of accurate predictive models of its rates of development at different temperatures. A laboratory colony of An. stephensi, collected from Bandar Abbas County, southern Iran, was established, and all its developmental stages were maintained in temperature-controlled incubators so that the water temperature set at 5, 8, 10, 12.5, 14, 28, 38, 39.5, 42, and 45(±0.2) °C for different treatments until subsequent adult emergence. The Lower and Upper Developmental Temperatures (LDT and UDT) and the growth degree-day (GDD) were calculated for each development stage. A 12-mo population dynamics survey of the larvae and adults of An. stephensi was performed in 3 malaria-endemic villages (Geno, Hormoodar, and Sarkhoon) of Bandar Abbas County, and the obtained data were matched with the constructed GDD model. Based on the field meteorological and dynamics data, the model was verified in the field and used to determine the appropriate date to start spraying. The LDT was determined to be 8.19, 9.74, 8.42, 5.6, 13.57, and 10.03 °C for egg hatching, first, second, and third ecdysis, pupation, and eclosion events, respectively. The UDT was 38 °C for all developmental stages. The thermal requirement for the development of all immature stages of An stephensi was determined to be 187.7 (±56.3) GDD above the LDT. Therefore, the appropriate date to start residual spraying is when the region's GDD reaches 187.7 (±56.3). Given the climatic conditions in Bandar Abbas County, it is expected that the first activity peak of adult An. stephensi would be in March. Field observations showed that An. stephensi activity starts in February and peaks in March. The GDD model can provide a good estimate for peak An. stephensi activity and indicate the optimal deployment time of residual spraying operations against the multiplication and development of malaria parasites inside the vector.

Pro-inflammatory cytokines are modified during the multiplication of Trypanosoma cruzi within the placental chorionic villi and are associated with the level of infection via the signaling pathway NF-κB.

Congenital Trypanosoma cruzi (T. cruzi) infection has been associated with changes in the levels of TNF-α and IFN-γ during the pregnancy. Therefore, we propose to study the participation and dynamics of proinflammatory cytokines in the infection process of placental explants infected by T. cruzi in vitro. Chorionic villous explants (CVE) obtained of human term placentas (n=8) from normal pregnancies were cultured with 105 trypomastigotes/mL of Tulahuen strain DTU VI for 0, 2, 4, 16, 24, 48 and 72h. Explants were treated with sulfasalazine (SULF) (5mM) and N-acetyl-cysteine (NAC) (15mM), as inhibitors molecules of NF-κB pathway, or LPS (1μg/mL) for 24 and 72h p.i. Motile trypomastigotes were counted in culture supernatants. Immunohistochemistry and ELISA for TNF-α, IFN-γ, IL-1β, IL-4, and IL-10 were performed in CVE and culture supernatants respectively. The parasite load was measured by RT-qPCR. T. cruzi invades the chorionic villi from 4h p.i. increasing significantly its DNA at 48 and 72h p.i. of culture (parasite multiplication phase). They were detected in stromal cells, which was related to elevation of TNF-α, IL-1β, IFN-γ, and IL-10. The inhibition of NF-κB activity in the explants decreased the production of the analyzed cytokines, showing elevated levels of T. cruzi DNA during the multiplication phase of the parasite. Placental tissue modifies the secretion of pro-inflammatory cytokines during the phase of parasite multiplication, but not during the invasion phase, which in turns modifies the level of infection via the signaling pathway NF-κB.

Intra-host Trypanosoma cruzi strain dynamics shape disease progression: the missing link in Chagas disease pathogenesis.

Chronic Chagasic cardiomyopathy develops years after infection in 20-40% of patients, but disease progression is poorly understood. Here, we assessed Trypanosoma cruzi parasite dynamics and pathogenesis over a 2.5-year period in naturally infected rhesus macaques. Individuals with better control of parasitemia were infected with a greater diversity of parasite strains compared to those with increasing parasitemia over time. Also, the in vivo parasite multiplication rate decreased with increasing parasite diversity, suggesting competition among strains or a stronger immune response in multiple infections. Significant differences in electrocardiographic (ECG) profiles were observed in Chagasic macaques compared to uninfected controls, suggesting early conduction defects, and changes in ECG patterns over time were observed only in macaques with increasing parasitemia and lower parasite diversity. Disease progression was also associated with plasma fibronectin degradation, which may serve as a biomarker. These data provide a novel framework for the understanding of Chagas disease pathogenesis, with parasite diversity shaping disease progression.IMPORTANCEChagas disease progression remains poorly understood, and patients at increased risk of developing severe cardiac disease cannot be distinguished from those who may remain asymptomatic. Monitoring of Trypanosoma cruzi strain dynamics and pathogenesis over 2-3 years in naturally infected macaques shows that increasing parasite diversity in hosts is detrimental to parasite multiplication and Chagasic cardiomyopathy disease progression. This provides a novel framework for the understanding of Chagas disease pathogenesis.

Tackling the emerging Artemisinin-resistant malaria parasite by modulation of defensive oxido-reductive mechanism via nitrofurantoin repurposing

Oxidative stress-mediated cell death has remained the prime parasiticidal mechanism of front line antimalarial, artemisinin (ART). The emergence of resistant Plasmodium parasites characterized by oxidative stress management due to impaired activation of ART and enhanced reactive oxygen species (ROS) detoxification has decreased its clinical efficacy. This gap can be filled by development of alternative chemotherapeutic agents to combat resistance defense mechanism. Interestingly, repositioning of clinically approved drugs presents an emerging approach for expediting antimalarial drug development and circumventing resistance. Herein, we evaluated the antimalarial potential of nitrofurantoin (NTF), a clinically used antibacterial drug, against intra-erythrocytic stages of ART-sensitive (Pf3D7) and resistant (PfKelch13R539T) strains of P. falciparum, alone and in combination with ART. NTF exhibited growth inhibitory effect at submicro-molar concentration by arresting parasite growth at trophozoite stage. It also inhibited the survival of resistant parasites as revealed by ring survival assay. Concomitantly, in vitro combination assay revealed synergistic association of NTF with ART. NTF was found to enhance the reactive oxygen and nitrogen species, and induced mitochondrial membrane depolarization in parasite. Furthermore, we found that exposure of parasites to NTF disrupted redox balance by impeding Glutathione Reductase activity, which manifests in enhanced oxidative stress, inducing parasite death. In vivo administration of NTF, alone and in combination with ART, in P. berghei ANKA-infected mice blocked parasite multiplication and enhanced mean survival time. Overall, our results indicate NTF as a promising repurposable drug with therapeutic potential against ART-sensitive as well as resistant parasites.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection.

There are no licensed vaccines against Plasmodium vivax. We conducted two phase 1/2a clinical trials to assess two vaccines targeting P. vivax Duffy-binding protein region II (PvDBPII). Recombinant viral vaccines using chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors as well as a protein and adjuvant formulation (PvDBPII/Matrix-M) were tested in both a standard and a delayed dosing regimen. Volunteers underwent controlled human malaria infection (CHMI) after their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparisons of parasite multiplication rates in the blood. PvDBPII/Matrix-M, given in a delayed dosing regimen, elicited the highest antibody responses and reduced the mean parasite multiplication rate after CHMI by 51% (n = 6) compared with unvaccinated controls (n = 13), whereas no other vaccine or regimen affected parasite growth. Both viral-vectored and protein vaccines were well tolerated and elicited expected, short-lived adverse events. Together, these results support further clinical evaluation of the PvDBPII/Matrix-M P. vivax vaccine.

Application of Silver Nanoparticles in Parasite Treatment.

Silver nanoparticles (AgNPs) are ultra-small silver particles with a size from 1 to 100 nanometers. Unlike bulk silver, they have unique physical and chemical properties. Numerous studies have shown that AgNPs have beneficial biological effects on various diseases, including antibacterial, anti-inflammatory, antioxidant, antiparasitic, and antiviruses. One of the most well-known applications is in the field of antibacterial applications, where AgNPs have strong abilities to kill multi-drug resistant bacteria, making them a potential candidate as an antibacterial drug. Recently, AgNPs synthesized from plant extracts have exhibited outstanding antiparasitic effects, with a shorter duration of use and enhanced ability to inhibit parasite multiplication compared to traditional antiparasitic drugs. This review summarizes the types, characteristics, and the mechanism of action of AgNPs in anti-parasitism, mainly focusing on their effects in leishmaniasis, flukes, cryptosporidiosis, toxoplasmosis, Haemonchus, Blastocystis hominis, and Strongylides. The aim is to provide a reference for the application of AgNPs in the prevention and control of parasitic diseases.

The Dantu blood group prevents parasite growth in vivo: Evidence from a controlled human malaria infection study.

The long co-evolution of Homo sapiens and Plasmodium falciparum has resulted in the selection of numerous human genetic variants that confer an advantage against severe malaria and death. One such variant is the Dantu blood group antigen, which is associated with 74% protection against severe and complicated P. falciparum malaria infections in homozygous individuals, similar to that provided by the sickle haemoglobin allele (HbS). Recent in vitro studies suggest that Dantu exerts this protection by increasing the surface tension of red blood cells, thereby impeding the ability of P. falciparum merozoites to invade them and reducing parasite multiplication. However, no studies have yet explored this hypothesis in vivo. We investigated the effect of Dantu on early phase P. falciparum (Pf) infections in a controlled human malaria infection (CHMI) study. 141 sickle-negative Kenyan adults were inoculated with 3.2 × 103 aseptic, purified, cryopreserved Pf sporozoites (PfSPZ Challenge) then monitored for blood-stage parasitaemia for 21 days by quantitative polymerase chain reaction (qPCR)analysis of the 18S ribosomal RNA P. falciparum gene. The primary endpoint was blood-stage P. falciparum parasitaemia of ≥500/μl while the secondary endpoint was the receipt of antimalarial treatment in the presence of parasitaemia of any density. On study completion, all participants were genotyped both for Dantu and for four other polymorphisms that are associated with protection against severe falciparum malaria: α+-thalassaemia, blood group O, G6PD deficiency, and the rs4951074 allele in the red cell calcium transporter ATP2B4. The primary endpoint was reached in 25/111 (22.5%) non-Dantu subjects in comparison to 0/27 (0%) Dantu heterozygotes and 0/3 (0.0%) Dantu homozygotes (p=0.01). Similarly, 49/111 (44.1%) non-Dantu subjects reached the secondary endpoint in comparison to only 7/27 (25.9%) and 0/3 (0.0%) Dantu heterozygotes and homozygotes, respectively (p=0.021). No significant impacts on either outcome were seen for any of the other genetic variants under study. This study reveals, for the first time, that the Dantu blood group is associated with high-level protection against early, non-clinical, P. falciparum malaria infections in vivo. Learning more about the mechanisms involved could potentially lead to new approaches to the prevention or treatment of the disease. Our study illustrates the power of CHMI with PfSPZ Challenge for directly testing the protective impact of genotypes previously identified using other methods. The Kenya CHMI study was supported by an award from Wellcome (grant number 107499). SK was supported by a Training Fellowship (216444/Z/19/Z), TNW by a Senior Research Fellowship (202800/Z/16/Z), JCR by an Investigator Award (220266/Z/20/Z), and core support to the KEMRI-Wellcome Trust Research Programme in Kilifi, Kenya (203077), all from Wellcome. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. NCT02739763.

A Plasmodium falciparum lysophospholipase regulates host fatty acid flux via parasite lipid storage to enable controlled asexual schizogony

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis of Plasmodium falciparum. To generate such phospholipids, the parasite extensively scavenges, recycles, and reassembles host lipids. P.falciparum possesses an unusually large number of lysophospholipases, whose roles and importance remain to be elucidated. Here, we functionally characterize one P.falciparum lysophospholipase, PfLPL3, to reveal its key role in parasite propagation during asexual blood stages. PfLPL3 displays a dynamic localization throughout asexual stages, mainly localizing in the host-parasite interface. Inducible knockdown of PfLPL3 disrupts parasite development from trophozoites to schizont, inducing a drastic reduction in merozoite progenies. Detailed lipidomic analyses show that PfLPL3 generates fatty acids from scavenged host lipids to generate neutral lipids. These are then timely mobilized to allow schizogony and merozoite formation. We then identify inhibitors of PfLPL3 from Medicine for Malaria Venture (MMV) with potent antimalarial activity, which could also serve as pertinent chemical tools to study parasite lipid synthesis.

Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Breeding honey bees (Apis mellifera L.) for low and high Varroa destructor population growth: Gene expression of bees performing grooming behavior.

Social organisms, including honey bees (Apis mellifera L.), have defense mechanisms to control the multiplication and transmission of parasites and pathogens within their colonies. Self-grooming, a mechanism of behavioral immunity, seems to contribute to restrain the population growth of the ectoparasitic mite Varroa destructor in honey bee colonies. Because V. destructor is the most damaging parasite of honey bees, breeding them for resistance against the mite is a high priority of the beekeeping industry. A bidirectional breeding program to select honey bee colonies with low and high V. destructor population growth (LVG and HVG, respectively) was conducted. Having high and low lines of bees allowed the study of genetic mechanisms underlying self-grooming behavior between the extreme genotypes. Worker bees were classified into two categories: 'light groomers' and 'intense groomers'. The brains of bees from the different categories (LVG-intense, LVG-light, HVG-intense, and HVG-light) were used for gene expression and viral quantification analyses. Differentially expressed genes (DEGs) associated with the LVG and HVG lines were identified. Four odorant-binding proteins and a gustatory receptor were identified as differentially expressed genes. A functional enrichment analysis showed 19 enriched pathways from a list of 219 down-regulated DEGs in HVG bees, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) term of oxidative phosphorylation. Additionally, bees from the LVG line showed lower levels of Apis rhabdovirus 1 and 2, Varroa destructor virus -1 (VDV-1/DWV-B), and Deformed wing virus-A (DWV-A) compared to bees of the HVG line. The difference in expression of odorant-binding protein genes and a gustatory receptor between bee lines suggests a possible link between them and the perception of irritants to trigger rapid self-grooming instances that require the activation of energy metabolic pathways. These results provide new insights on the molecular mechanisms involved in honey bee grooming behavior. Differences in viral levels in the brains of LVG and HVG bees showed the importance of investigating the pathogenicity and potential impacts of neurotropic viruses on behavioral immunity. The results of this study advance the understanding of a trait used for selective breeding, self-grooming, and the potential of using genomic assisted selection to improve breeding programs.

Senna occidentalis (L.) Link root extract inhibits Plasmodium growth in vitro and in mice

BackgroundSenna occidentalis (L.) Link has been used worldwide in traditional treatment of many diseases and conditions including snakebite. In Kenya, a decoction from the plant roots taken orally, is used as a cure for malaria. Several studies have demonstrated that extracts from the plant possess antiplasmodial activity, in vitro. However, the safety and curative potency of the plant root against established malaria infection is yet to be scientifically validated, in vivo. On the other hand, there are reports on variation in bioactivity of extracts obtained from this plant species, depending on the plant part used and place of origin among other factors. In this study, we demonstrated the antiplasmodial activity of Senna occidentalis roots extract in vitro, and in mice.MethodsMethanol, ethyl acetate, chloroform, hexane and water extracts of S. occidentalis root were tested for in vitro antiplasmodial activity against Plasmodium falciparum, strain 3D7. Cytotoxicity of the most active solvent extracts was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the curative potency in Plasmodium berghei infected mice evaluated by Rane’s test.ResultsAll of the solvent extracts tested in this study inhibited the propagation of P. falciparum, strain 3D7, in vitro, with polar extracts being more active than non-polar ones. Methanolic extracts had the highest activity (IC50 = 1.76) while hexane extract displayed the lowest activity (IC50 = 18.47). At the tested concentrations, methanolic and aqueous extracts exhibited high selectivity index against P. falciparum strain 3D7 (SI > 10) in the cytotoxicity assay. Further, the extracts significantly suppressed the propagation of P. berghei parasites (P < 0.05) in vivo and increased the survival time of the infected mice (P < 0.0001).ConclusionsSenna occidentalis (L.) Link root extract inhibits the propagation of malaria parasites in vitro and in BALB/c mice.

Past, current, and potential treatments for cryptosporidiosis in humans and farm animals: A comprehensive review.

The intracellular protozoan parasite of the genus Cryptosporidium is among the leading causes of waterborne diarrheal disease outbreaks throughout the world. The parasite is transmitted by ingestion of infective oocysts that are highly stable in the environment and resistant to almost all conventional disinfection methods and water treatments. Control of the parasite infection is exceedingly difficult due to the excretion of large numbers of oocysts in the feces of infected individuals that contaminate the environment and serve as a source of infection for susceptible hosts including humans and animals. Drug development against the parasite is challenging owing to its limited genetic tractability, absence of conventional drug targets, unique intracellular location within the host, and the paucity of robust cell culture platforms for continuous parasite propagation. Despite the high prevalence of the parasite, the only US Food and Drug Administration (FDA)-approved treatment of Cryptosporidium infections is nitazoxanide, which has shown moderate efficacy in immunocompetent patients. More importantly, no effective therapeutic drugs are available for treating severe, potentially life-threatening cryptosporidiosis in immunodeficient patients, young children, and neonatal livestock. Thus, safe, inexpensive, and efficacious drugs are urgently required to reduce the ever-increasing global cryptosporidiosis burden especially in low-resource countries. Several compounds have been tested for both in vitro and in vivo efficacy against the disease. However, to date, only a few experimental compounds have been subjected to clinical trials in natural hosts, and among those none have proven efficacious. This review provides an overview of the past and present anti-Cryptosporidium pharmacotherapy in humans and agricultural animals. Herein, we also highlight the progress made in the field over the last few years and discuss the different strategies employed for discovery and development of effective prospective treatments for cryptosporidiosis.

A Plasmodium falciparum ubiquitin-specific protease (PfUSP) is essential for parasite survival and its disruption enhances artemisinin efficacy.

Proteins associated with ubiquitin-proteasome system (UPS) are potential drug targets in the malaria parasite. The ubiquitination and deubiquitination are key regulatory processes for the functioning of UPS. In this study, we have characterized the biochemical and functional role of a novel ubiquitin-specific protease (USP) domain-containing protein of the human malaria parasite Plasmodium falciparum (PfUSP). We have shown that the PfUSP is an active deubiquitinase associated with parasite endoplasmic reticulum (ER). Selection linked integration (SLI) method for C-terminal tagging and GlmS-ribozyme mediated inducible knock-down (iKD) of PfUSP was utilized to assess its functional role. Inducible knockdown of PfUSP resulted in a remarkable reduction in parasite growth and multiplication; specifically, PfUSP-iKD disrupted ER morphology and development, blocked the development of healthy schizonts, and hindered proper merozoite development. PfUSP-iKD caused increased ubiquitylation of specific proteins, disrupted organelle homeostasis and reduced parasite survival. Since the mode of action of artemisinin and the artemisinin-resistance are shown to be associated with the proteasome machinery, we analyzed the effect of dihydroartemisinin (DHA) on PfUSP-iKD parasites. Importantly, the PfUSP-knocked-down parasite showed increased sensitivity to dihydroartemisinin (DHA), whereas no change in chloroquine sensitivity was observed, suggesting a role of PfUSP in combating artemisinin-induced cellular stress. Together, the results show that Plasmodium PfUSP is an essential protease for parasite survival, and its inhibition increases the efficacy of artemisinin-based drugs. Therefore, PfUSP can be targeted to develop novel scaffolds for developing new antimalarials to combat artemisinin resistance.

Buffalo colostrum- A novel substitute of human serum for the cultivation of Plasmodium falciparum in vitro

In vitro cultivation of erythrocytic stages of Plasmodium falciparum requires supplementing the culture medium with human serum. The present study was carried out to explore an alternative to human serum. Different human serum samples were found to vary considerably in their ability to support the growth of erythrocytic stages of P. falciparum in vitro. These results strongly suggested the use of pooled human serum for comparing the growth of parasites in medium augmented with other supplements. Parasites could multiply for a few cycles only in RPMI (Roswell Park Memorial Institute) medium supplemented with serum obtained from pig, goat, sheep or buffalo. Continued cultivation could not be achieved using any one of these animal sera. Ability of bovine colostrum was investigated as an alternative to human serum. Buffalo colostrum, 10%(v/v) 'suitably prepared' supported the continuous growth and multiplication of P. falciparum . Morphologically both asexual and sexual stages appeared normal and healthy, but the multiplication rate of parasites grown in colostrum augmented medium was found to be lower than that in serum-supplemented medium. The one month of uninterrupted cultivation of P. falciparum registered 106 fold increase in parasite density compared to 1010 fold multiplication recorded in control culture with 10% serum supplement. Cow colostrum failed to support the growth and multiplication of parasites beyond 6 days in culture. The initial positive results with buffalo colostrum hold promise and should be explored further as a potential substitute for human serum for continuous in vitro propagation of erythrocytic stages of the malignant malaria parasite.