Recognition with monoclonal antibodies of a Mr 71000 surface antigen of Plasmodium falciparum merozoites

Merozoite surface antigens can induce protective immune responses and may becandidate antigens for malaria vaccine.Four hybridoma cell lines secreting monoclonalantibodies against Plasmodium falciparum Fcc7801/HN were produced.Antibodies fromthree of the four lines showed significant growth-inhibiting effect on P.falciparum invitro.One monoclonal antibody,known as C6,conjugated the antigen located exclusivelyon the merozoite surface and distributed evenly over the entire surface,as wasdemonstrated by immunoelectron microscopy.C6 also precipitated a single protein of Mr71000.

Andrographolide effect on both Plasmodium falciparum infected and noninfected RBCs membranes

Objective: To explore whether its antiplasmodium effect of andrographolide is attributed to its plausible effect on the plasma membrane of both Plasmodium falciparum infected and noninfected RBCs. Methods: Anti-plasmodium effect of andrographolide against Plasmodium falciparum strains was screened using the conventional malaria drug sensitivity assay. The drug was incubated with uninfected RBCs to monitor its effect on their morphology, integrity and osmotic fragility. It was incubated with the plasmodium infected RBCs to monitor its effect on the parasite induced permeation pathways. Its effect on the potential of merozoites to invade new RBCs was tested using merozoite invasion assay. Results: It showed that at andrographolide was innocuous to RBCs at concentrations approach its therapeutic level against plasmodia. Nevertheless, this inertness was dwindled at higher concentrations. Conclusions: In spite of its success to inhibit plasmodium induced permeation pathway and the potential of merozoites to invade new RBCs, its anti-plasmodium effect can't be attributed to these functions as they were attained at concentrations higher than what is required to eradicate the parasite. Consequently, other mechanisms may be associated with its claimed actions.

Lactate dehydrogenase:An important molecule involved in acetamizuril action against Eimeria tenella

Lactate dehydrogenase(LDH),a vital enzyme in anaerobic glycolysis,is closely associated with the survival of parasites.Previous studies of some parasites have shown that LDH exhibits unique physicochemical properties and molecular structures and may be an ideal potential target for diagnosis and drug screening.In this study,we aimed to investigate the effects of acetamizuril,a novel anticoccidial compound,on LDH in the second-generation merozoites of Eimeria tenella(mz-LDH).Quantitative real-time PCR,Western blot,immunofluorescence and enzyme activity assays were each applied to detect the changes of mz-LDH.Our results indicated that the mRNA and protein levels of mz-LDH were reduced upon acetamizuril treatment.Immunolocalization of mz-LDH demonstrated that considerable amount of mz-LDH was distributed around or in the nuclei of second-generation merozoites within the untreated group;in contrast,the acetamizuril-treated group had very low level of mz-LDH.Meanwhile,LDH enzyme activity assay suggested that a decreased LDH enzyme activity in both cytoplasm and nucleus of merozoites in the acetamizuril-treated group.Moreover,the induced apoptosis in second-generation merozoites by the acetamizuril was evaluated by detecting caspase 3 activity,and acetamizuril was found to significantly increase caspase 3 activity.The above findings show that LDH may play an important role in the mediating the activity of acetamizuril against coccidiosis,and further investigation into this aspect might contribute to new light on the pathogenesis of E.tenella during its interaction with acetamizuril.

MIC17A is a novel diagnostic marker for feline toxoplasmosis

Toxoplasma gondii is a widespread parasitic pathogen that infect humans and all warm-blooded animals,causing abortion and stillbirth in pregnant women and animals,as well as life threatening toxoplasmosis in immune compromised individuals.Felines are the only defnitive hosts of Toxoplasma and oocysts shed by infected felines are the major source of infection for humans and other animals.Given the critical role of felines for T.gondii transmission,control of feline toxoplasmosis has signifcant impacts on reducing the overall prevalence of animal and human toxoplasmosis.However,reliable diagnosis of feline toxoplasmosis is still challenging.In this study,we found that the putative micronemal protein 17A(MIC17A)that was abundantly expressed in Toxoplasma merozoites is a good diagnostic marker for serological diagnosis of Toxoplasma infection in felines.T.gondii encodes four paralogs of MIC17A in total and the expression of three of them is drastically upregulated in merozoites than in tachyzoites.In contrast,when proteins like GRA1 and MIC3 that are more abundantly expressed in tachyzoites than in merozoites were used as diagnostic antigens to test feline toxoplasmosis,they reacted with Toxoplasma specifc IgG antibodies poorly.Taken together,these results suggest that merozoite antigens are better suited for the diagnosis of feline toxoplasmosis than antigens that are highly expressed at tachyzoite or bradyzoite stages.

Limited genetic diversity among Plasmodium falciparium isolates using nested PCR in Jazan Area, Saudi Arabia

Objective: To establish molecular characterization of Plasmodium falciparum field isolates in Jazan Area of Saudi Arabia measured with highly polymorphic genetic marker, i.e. the merozoite surface protein 2(MSP 2).Methods: Blood samples were collected from 128 clinically suspected patients attending both Jazan and Sabia hospitals, Kingdom of Saudi Arabia. Both hospitals reflected urban and rural settings respectively. Analysis of central polymorphic region of MSP 2(3D7 and FC27 allelic families) was performed using nested PCR for malaria patients.Results: For MSP 2 allelic families of Plasmodium falciparum, 16 cases(53.3%) carried FC27 type and 14 cases(46.7%) carried 3D7 type, whereas no malaria cases harbored both allelic types. The present study showed that in urban area, 80% of FC27 fragments were 500 bp while in rural area it was 45.5%(P = 0.08). The FC27 400 bp allele was more prevalent in patients from rural than those from the urban area(P = 0.08). The most prevalent infecting 3D7 allele was the 3D7 300 bp in both areas. In the present study, there were no multiple infections.Conclusions: The limited genetic diversity which was observed in Jazan(considered as an endemic area) may be attributed to the small sample size or sustained malaria control program.

Cocktail of Theileria equi antigens for detecting infection in equines

Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain,Theileria equi(T.equi)protein 82(Te 82)and T.equi 104 k Da microneme-rhoptry antigen precursor(Te 43),to diagnose T.equi infection in horses as compared with equi merozoite antigen-2(EMA-2).Methods:In the current study,we applied a cocktail-ELISA containing two antigens(EMA-2+Te 82)to diagnose T.equi infection either in experimentally infected horses or in field infection.Results:Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T.equi infection in horses as compared with Te 82 or Te 43 alone.Conclusions:The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

Subcloning,sequencing and expressing of conserved blocks of P190 gene of Plasmodium falciparum FCC1/HN strain

190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according to the sequence of MAD20 strain,with a GCclamp and BamH Ⅰ site at the 5’-end of each one,and a GC clamp and Xba Ⅰ site at the 3’-endof each one.The primers were synthesized by phosphoramidite approach (User’s Manual ofABI Company) and purified using HPLC.Three fragments in the second,third and fourth con-served regions of P190 gene of Plasrnodium falciparum FCC1/HN strain isolated from theblood of patients in Hainan Province of China were amplified by the polymerase chain reaction(PCR).The amplified fragments were suhcloned into pUC18 vectors and sequenced using thedideoxy chain termination method.All three regions of P190 gene of FCC1/HN strain also werehighly conservative as compared with P190 gene of MAD20 (Papua New Guinea isolate),K1(Thailand isolate),Wellcome (West Africa isolate) and CAMP (Malaysia) strains ofPlasmodium falciparum.The C at position 81 in the second conserved block of P190 gene ofFCC1/HN isolate was substituted by T,which did not change the amino acid determined by thecodon corresponding to the substitution.The genes sequenced were cloned into rpGEX-2T,aglutathione S-transferase gene fusion system for expression.

In-Host Analysis of Malaria Dynamics in Humans

Human malaria infection poses a major global health threat worldwide. Yet, no sophisticated mathematical model exists to study the complex dynamics and interactions between the parasites and host immune response at the blood and liver stages. In this paper, an in-host mathematical model of Plasmodium falciparum malaria dynamics and interactions in an infected host cells are studied at the liver stage by incorporating the red blood cells and the immune system. Numerical simulations are applied to investigate the interactions between the host immune response, the parasite dynamics, and the disease dynamics at both the blood and liver stages. Results show that immunity has a significant impact in clearing infected red blood cells. Furthermore, the infected erythrocytes and hence the severity of malaria tend to increase with increasing density of merozoites in the blood. The result revealed that intervention during malaria infection should focus on minimizing merozoite invasion rate on healthy erythrocytes and the density of merozoites in circulation.

Immune evasion by Plasmodium falciparum parasites: converting a host protection mechanism for the parasite′s benefit

Immune evasion is a strategy used by pathogenic microbes to evade the host immune system in order to ensure successful propagation. Immune evasion is particularly important for the blood stages of Plasmodium falciparum, the causative agent of the deadly disease malaria tropica. Because Plasmodium blood stage parasites require human erythrocytes for replication, their ability to evade attack by the human immune system is essential for parasite survival. In order to escape immunity-induced killing, the intraerythrocytic parasites have evolved a variety of evasion mechanisms, including expansion of plasmodial surface proteins, organ-specific sequestration of the infected red blood cells and acquisition of immune-regulatory proteins by the parasite. This review aims to highlight recent advances in the molecular understanding of the immune evasion strategies by P. falciparum, including antigenic variation, surface protein polymorphisms and invasion ligand diversification. The review will further discuss new findings on the regulatory mechanisms applied by P. falciparum to avoid lysis by the human complement as well as killing by immune factors of the mosquito vector.