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A rapid sporozoite ELISA using 3,3',5,5'-tetramethylbenzidine as the substrate chromogen

Lee M, Harrison BA, Lewis GE

Am J Trop Med Hyg, 1990 Apr;42(4):314-9.
PMID: 2184690 DOI: 10.4269/ajtmh.1990.42.314

A modified version of the standard 2-site sporozoite enzyme-linked immunosorbent assay (ELISA) using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate chromogen solution was adapted for rapid detection and identification of Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins. The TMB-ELISA was evaluated using sporozoites from experimentally infected mosquitoes and laboratory colonized uninfected mosquitoes. Our data indicate comparable sensitivity levels between the TMB-ELISA and the standard ELISA, i.e., 50 P. falciparum or P. vivax sporozoites/50 microliters of test solution. Reactions inherent to the method were specific and background reactivity was minimal. The TMB-ELISA is rapid (1 hr), simple, uses a minimal amount of monoclonal antibodies, and is suitable for use in a wide range of laboratories.

Matched MeSH terms: Antigens, Protozoan/analysis*
Fulltext Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum

Matsubayashi M, Teramoto-Kimata I, Uni S, Lillehoj HS, Matsuda H, Furuya M, et al.

J Biol Chem, 2013 Nov 22;288(47):34111-34120.
PMID: 24085304 DOI: 10.1074/jbc.M113.515544

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Matched MeSH terms: Antigens, Protozoan/genetics; Antigens, Protozoan/immunology*; Antigens, Protozoan/metabolism
Fulltext Induction and Kinetics of Complement-Fixing Antibodies Against Plasmodium vivax Merozoite Surface Protein 3α and Relationship With Immunoglobulin G Subclasses and Immunoglobulin M

Oyong DA, Wilson DW, Barber BE, William T, Jiang J, Galinski MR, et al.

J Infect Dis, 2019 11 06;220(12):1950-1961.
PMID: 31419296 DOI: 10.1093/infdis/jiz407

BACKGROUND: Complement-fixing antibodies are important mediators of protection against Plasmodium falciparum malaria. However, complement-fixing antibodies remain uncharacterized for Plasmodium vivax malaria. P. vivax merozoite surface protein 3α (PvMSP3α) is a target of acquired immunity and a potential vaccine candidate.
METHODS: Plasma from children and adults with P. vivax malaria in Sabah, Malaysia, were collected during acute infection, 7 and 28 days after drug treatment. Complement-fixing antibodies and immunoglobulin M and G (IgM and IgG), targeting 3 distinctive regions of PvMSP3α, were measured by means of enzyme-linked immunosorbent assay.
RESULTS: The seroprevalence of complement-fixing antibodies was highest against the PvMSP3α central region (77.6%). IgG1, IgG3, and IgM were significantly correlated with C1q fixation, and both purified IgG and IgM were capable of mediating C1q fixation to PvMSP3α. Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults. Levels of functional antibodies increased after acute infection through 7 days after treatment but rapidly waned by day 28.
CONCLUSION: Our study demonstrates that PvMSP3α antibodies acquired during P. vivax infection can mediate complement fixation and shows the important influence of age in shaping these specific antibody responses. Further studies are warranted to understand the role of these functional antibodies in protective immunity against P. vivax malaria.

Matched MeSH terms: Antigens, Protozoan/immunology*
Fulltext Low Levels of Polymorphisms and Negative Selection in Plasmodum knowlesi Merozoite Surface Protein 8 in Malaysian Isolates

Atique Ahmed M, Kang HJ, Quan FS

Korean J Parasitol, 2019 Aug;57(4):445-450.
PMID: 31533414 DOI: 10.3347/kjp.2019.57.4.445

Human infections due to the monkey malaria parasite Plasmodium knowlesi is increasingly being reported from most Southeast Asian countries specifically Malaysia. The parasite causes severe and fatal malaria thus there is a need for urgent measures for its control. In this study, the level of polymorphisms, haplotypes and natural selection of full-length pkmsp8 in 37 clinical samples from Malaysian Borneo along with 6 lab-adapted strains were investigated. Low levels of polymorphism were observed across the full-length gene, the double epidermal growth factor (EGF) domains were mostly conserved, and non-synonymous substitutions were absent. Evidence of strong negative selection pressure in the non-EGF regions were found indicating functional constrains acting at different domains. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. This is the first study to genetically characterize the full-length msp8 gene from clinical isolates of P. knowlesi from Malaysia; however, further functional characterization would be useful for future rational vaccine design.

Matched MeSH terms: Antigens, Protozoan/genetics*; Antigens, Protozoan/immunology
Fulltext Seroprevalence of Sarcocystis falcatula in Two Islands of Malaysia using Recombinant Surface Antigen 4

Nadzirah TTI, Yik FM, Ling LY

Korean J Parasitol, 2020 Feb;58(1):1-5.
PMID: 32145721 DOI: 10.3347/kjp.2020.58.1.1

Sarcocystosis was diagnosed worldwide by serodiagnostic tests utilising the whole parasite, for which the protozoa were maintained in vitro are more costly. In this study, antigenicity of Sarcocystis falcatula recombinant protein (rSfSAG4) was investigated towards the local communities of Pangkor and Tioman Islands and its seroprevalence was surveyed in these islands. A total of 348 human sera were tested using rSfSAG4 by Western blot and ELISA. High prevalence of sarcocystosis was observed in Tioman Island (80.6%) than in Pangkor Island (50.0%) by Western blot. In ELISA, the seroprevalence observed in Tioman Island was 45.9%, whereas in Pangkor Island 63.0%. In other parasitic infections, the prevalence was 34.0% by Western blot and 46.0% by ELISA. In healthy control group, 7% by Western blot and 8% by ELISA showed positivity to rSfSAG4. It is suggested SfSAG4 is a candidate antigen to measure seroprevalence of sarcocystosis.

Matched MeSH terms: Antigens, Protozoan/immunology*
DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice

Maspi N, Ghaffarifar F, Sharifi Z, Dalimi A, Khademi SZ

Malays J Pathol, 2017 Dec;39(3):267-275.
PMID: 29279589

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levels compared to control groups (p<0.05). IFN-γ/ Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstrated the highest IFN-γ and IgG2a levels in the group receiving LACK-TSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (p<0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA and TSA groups than LACK group after challenge (p<0.05). In the present study, DNA immunization promoted Th1 immune response and confirmed the previous observations on immunogenicity of LACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major.

Matched MeSH terms: Antigens, Protozoan/immunology*
Cloning and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris

Lau YL, Fong MY, Idris MM, Ching XT

Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):10-6.
PMID: 23082548

Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZalpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of -28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.

Matched MeSH terms: Antigens, Protozoan/genetics*; Antigens, Protozoan/metabolism
Optimization for high-level expression in Pichia pastoris and purification of truncated and full length recombinant SAG2 of Toxoplasma gondii for diagnostic use

Ling LY, Ithoi I, Yik FM

Southeast Asian J. Trop. Med. Public Health, 2010 May;41(3):507-13.
PMID: 20578535

SAG2 is one of the major surface antigens of the intracellular protozoan parasite Toxoplasma gondii. In the present study, truncated recombinant SAG2(S) and full length recombinant SAG2(T) of T. gondii were optimally produced (approximately 15 mg/liter) in Pichia pastoris expression system using BMMY medium at pH 3, 25 degrees C in 0.5-1% methanol and a time-course of 1-2 days. The recombinant proteins were purified using a commercial gel filtration purification system obtaining approximately 33% recovery. The purified SAG2(S) and SAG2(T) showed molecular masses of 45 and 36 kDa by SDS-PAGE, respectively. The recombinant proteins were evaluated by Western blotting with patients' sera and demonstrated 90% sensitivity and 100% specificity for detection of toxoplasmosis. This study provided a means for large-scale expression and purification of SAG2, which should be useful for diagnosis of toxoplasmosis.

Matched MeSH terms: Antigens, Protozoan/biosynthesis*
Fulltext Serological evaluation of malaria patients in Thailand: antibody response against electrophoresed antigenic polypeptides of Plasmodium falciparum

Kano S, Onda T, Matsumoto Y, Buchachart K, Krudsood S, Looareesuwan S, et al.

Southeast Asian J. Trop. Med. Public Health, 1998 Jun;29(2):341-3.
PMID: 9886125

It was reported that a 47kDa antigenic polypeptide of Plasmodium falciparum had been strongly presented by the sera from 1) imported Japanese malaria patients with severe symptoms and 2) symptomatic and parasitemic inhabitants in endemic areas in the Sudan, Malaysia and the Philippines. In the present study, we observed the reactivity of the sera from falciparum malaria patients who had been hospitalized in the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, and compared the antibody response against the 47kDa antigenic polypeptide according to the severity of the patients. It was observed that antibodies to this molecule were more commonly shared in sera from severer patients, although the IFAT titers against the whole P. falciparum parasite antigen were lower in the group, which suggested that this antibody against the 47kDa molecule was playing a specific role at a severe stage of the infection. Determination of the immunological features of the antigenic molecules of parasites by this type of sero-epidemiological study will provide a new assay system for evaluation of immune status of individuals in different severity and suggest a way of vaccine development.

Matched MeSH terms: Antigens, Protozoan/immunology*
Fulltext Detection of circulating plasmodial antigens in human sera by sandwich ELISA with monoclonal antibodies

Lim PK, Mak JW, Yong HS

Southeast Asian J. Trop. Med. Public Health, 1992 Dec;23(4):735-9.
PMID: 1298082

Two monoclonal antibodies (MAbs), one produced against Plasmodium falciparum (PF-IG8) and the other against P. cynomolgi (PC-IE12) schizont antigens were used in a sandwich ELISA for the detection of circulating plasmodial antigens in sera of patients infected with either P. falciparum, P. vivax or P. malariae. The mean +/- SD optical density (OD) values for the normal control group using PF-108 and PC-1E12 were 0.351 +/- 0.036 and 0.205 +/- 0.044, respectively. Mean OD values for the three infected groups were found to be significantly higher than those of the normal control group for both MAbs. However, ELISA values for individual serum specimens did not correlate with the level of parasitemia in the infected blood. Using a cut-off point of mean OD +/- 3 SD of the normal control group as indicating a positive reading, the specificity of this assay with both MAbs was 100%. The sensitivity of the assay using PF-1G8 was 95% while that obtained with PC-1E12 was 98%.

Matched MeSH terms: Antigens, Protozoan/blood*
Long term maintenance of Toxoplasma gondii (Rh strain) in Vero cell line and use of harvested antigens for immunodiagnosis

Suresh K, Mak JW, Yong HS

Southeast Asian J. Trop. Med. Public Health, 1991 Dec;22 Suppl:124-8.
PMID: 1822869

Thirty in vitro serial passages of Toxoplasman gondii cultures in Vero cell line performed once in every five days had a mean increase in parasite count of 74.4 +/- 14.8 times from that of initial counts. Long term cultures in Vero cell line did not alter the virulence of the parasite. The good correlation (r = 0.99) between the IFA titer and ELISA OD values using the parasite antigens from in vitro sources indicates that long term maintenance of T. gondii in culture does not affect significantly the ability to recognize antibodies to surface and soluble antigens. The results also show that soluble antigens containing host cells can be directly used for immunodiagnostic purposes without purification. The in vitro maintenance of T. gondii is safer and cheaper when compared to the in vivo method.

Matched MeSH terms: Antigens, Protozoan*
Fulltext Immune response in acute Toxoplasma infection of Balb/C, ICR and CBA/J mice

Suresh K, Mak JW, Yong HS

Southeast Asian J. Trop. Med. Public Health, 1991 Sep;22(3):452-4.
PMID: 1818401
Matched MeSH terms: Antigens, Protozoan/immunology
Fulltext Efficacies of two in-house indirect ELISAs for diagnosis of amoebic liver abscess

Tan ZN, Wong WK, Noordin R, Zeehaida M, Olivos GA, Lim BH

Trop Biomed, 2013 Jun;30(2):250-6.
PMID: 23959490 MyJurnal

Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.

Matched MeSH terms: Antigens, Protozoan
Fulltext Immunogenicity of recombinant BCG-based vaccine expressing the 22 kDa of serine repeat antigen (SE22) of Plasmodium falciparum

Teo WH, Nurul AA, Norazmi MN

Trop Biomed, 2012 Jun;29(2):239-53.
PMID: 22735846 MyJurnal

The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.

Matched MeSH terms: Antigens, Protozoan/genetics; Antigens, Protozoan/immunology*
Fulltext Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris

Thiruvengadam G, Init I, Fong MY, Lau YL

Trop Biomed, 2011 Dec;28(3):506-13.
PMID: 22433878 MyJurnal

Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

Matched MeSH terms: Antigens, Protozoan/biosynthesis*; Antigens, Protozoan/genetics; Antigens, Protozoan/isolation & purification
Fulltext Determination of the specificities of monoclonal and polyclonal antibodies to Neospora, Toxoplasma and Cryptosporidium by fluorescent antibody test (FAT)

Latif BM, Jakubek EB

Trop Biomed, 2008 Dec;25(3):225-31.
PMID: 19287361

Flourescent antibody test (FAT) was applied to determine the cross-reactivities of monoclonal (mAb), polyclonal (pAb) antibodies to Neospora, Toxoplasma and Cryptosporidium and antisera from cattle naturally infected with Neospora canium against antigens from a number of sources. Both mAb and pAb to Neospora reacted strongly (FAT titre up to 2560) with the homologous antigens and demonstrated weak titre (80) or no reaction with both Toxoplasma and Cryptosporidium antigens. Also mAb and pAb to Toxoplasma gondii reacted at titres of 80 - 640 with homologous antigens and at titres of 10-40 with N. caninum. No cross-reactions with either mAb or pAb antibodies to N. caninum and T. gondii were observed with Cryptosporidium parvum. The same results were observed with C. parvum mAb when tested with both N. caninum and T. gondii antigens. Sera from cattle naturally infected with N. caninum had titres ranging from 80- 640 with N. caninum antigens, and 10- 40 with T. gondii and C. parvum antigens. At low dilutions, the complete surfaces of Neospora and Toxoplasma parasites were fluorescent, while in higher dilutions only dotted fluorescence appeared on the apical complex. These results indicated the presence of cross-reactivity between Neospora and Toxoplasma but not with Cryptosporidium. Accordingly the recommended cut-off antibody titre for diagnosis of neosporosis is 80.

Matched MeSH terms: Antigens, Protozoan/immunology*
Fulltext A study on the usefulness of Techlab Entamoeba histolytica II antigen detection ELISA in the diagnosis of amoebic liver abscess (ALA) at Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia

Zeehaida M, Wan Nor Amilah WA, Amry AR, Hassan S, Sarimah A, Rahmah N

Trop Biomed, 2008 Dec;25(3):209-16.
PMID: 19287359

Amoebic serodiagnosis at Hospital Universiti Sains Malaysia (HUSM), Kelantan employs an indirect haemagglutination assay (IHA) which detects anti-Entamoeba histolytica antibodies in patients' serum samples. In an amoebiasis endemic area such as Kelantan, interpretation of a positive IHA result can be problematic due to the high background antibody levels. The TechLab E. histolytica II ELISA is a commercial kit for detection of specific Gal/GalNAc lectin antigen in stool samples, and has been reported to be able to detect the antigen in serum samples from patients with amoebic liver abscess (ALA). Thus in this study we investigated the usefulness of TechLab E. histolytica II ELISA for diagnosis of ALA by comparing it with IHA. This is a cross sectional study involving 58 suspected ALA patients who were admitted to the surgical ward, HUSM, Kelantan. The diagnosis of ALA was established based on clinical symptoms and signs, ultrasound and/or CT scan results. The serum specimens obtained from the patients were tested with IHA (Dade Behring Diagnostics, Marburg, Germany) and TechLab E. histolytica II ELISA (Techlab, Blacksburg, Virginia, USA) according to the manufacturers' instructions. Of the 58 patients, 72.4% (42) were positive by IHA and only 8.6% (5) were positive by the TechLab E. histolytica II ELISA. Agreement between the IHA and ELISA was poor (kappa value 0.019, p=0.691). There was also no correlation between ELISA results and IHA antibody titers. The TechLab E. histolytica II ELISA was not sensitive in detecting amoebic antigen in samples from ALA patients. In addition the results of the test did not correlate with the IHA anti-E. histolytica antibody titres. Therefore, the TechLab E. histolytica II ELISA was found not to be useful for serological diagnosis of ALA at HUSM.

Matched MeSH terms: Antigens, Protozoan/blood*
Fulltext Characterisation of a truncated Toxoplasma gondii surface antigen 2 (SAG2) secreted by the methylotrophic yeast Pichia pastoris

Lau YL, Shamilah H, Fong MY

Trop Biomed, 2006 Dec;23(2):186-93.
PMID: 17322821 MyJurnal

A truncated form of surface antigen 2 (SAG2) of the protozoan parasite Toxoplasma gondii was cloned and expressed in the methylotrophic yeast Pichia pastoris. This recombinant antigen, designated as recSAG2-N, contained only the N-terminal half of the native SAG2. The recSAG2-N was secreted by the Pichia pastoris into the culture supernatant, and it was harvested by using the trichloroacetic acid precipitation method. Specificity of recSAG2-N was evaluated in western blot assays. Fifty human serum samples, including 32 from confirmed cases of toxoplasmosis, were tested. Results from the assays showed that recSAG2-N reacted with sera from the toxoplasmosis cases only. In vivo experiments showed that serum from mice which received recSAG2-N reacted with the native SAG2 of T. gondii.

Matched MeSH terms: Antigens, Protozoan/metabolism*
Identification and characterization of Leishmania amastigote and axenic form antigens

Abdelhaleem AA, Elamin EM, Bakheit SM, Mukhtar MM

Trop Biomed, 2019 Dec 01;36(4):866-873.
PMID: 33597459

This study was aimed to identify and characterize Leishmania amastigote, and axenic form antigens. Two in vitro techniques were used to change leishmania parasite isolates from promastigote form to amastigotes and amastigote like (axenic) forms. The main strategy relied upon in vitro infection of murine macrophages cell line J774 with leishmania promastigote, at 37°C with 5% CO2, while the second technique relied upon the culture of promastigote at 37°C with low pH (5.5), and 5-10% CO2. Proteins were extracted and fractionated utilizing 12% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE). Antigens were recognized using both immune dot blot and western blot procedures. PCR was performed for recognition of leishmania parasites in infected J774 macrophages. L. major was quicker in infectivity of macrophages cell line than L. donovani. Shared proteins ranging from 26-116 kDa were identified by SDS PAGE in all stages. Immune Dot-blot method showed positive outcomes, while western blot identified an exceptional antigen band of 16 kDa in amastigote, this unique band could be of value in diagnosis and vaccination of leishmaniasis. PCR results confirmed presence of both isolates demonstrating that coinfection is conceivable, and no indications of hereditary recombination at kinetoplast DNA (kDNA) were identified in macrophages simultaneously infected by L. major and L. donovani.

Matched MeSH terms: Antigens, Protozoan/analysis*
Epitope variances demonstrated by Blastocystis sp. ST3 symptomatic and asymptomatic isolates

Sheela DS, Chandramathi S, Suresh K

Trop Biomed, 2020 Mar 01;37(1):210-217.
PMID: 33612732

Blastocystis sp. is an enteric protozoan parasite of humans and many animals. Blastocystis sp. subtype 3 (ST3) proves to be the highest frequency case in most populations around the world and it is further distinguished into symptomatic and asymptomatic isolates based on the clinical symptoms exhibited by infected individuals. Phenotypic and genotypic studies implicate the distinctiveness of this parasite which may describe its pathogenesis. However, the antigenic distinctiveness which describes the antibody mediated cell lysis of this parasite has not been explored. This study was aimed to identify the cross-reactivity and cytotoxicity effect between three isolates of symptomatic and asymptomatic Blastocystis sp. ST3 respectively. Antigen specificity and diversity of this parasite was performed by coculturing sera (10-fold dilution) obtained from mice immunised with Blastocystis sp. symptomatic and asymptomatic antigens and the respective Blastocystis sp. ST3 live cells through complement dependant cell cytotoxicity (CDC) assay. The results obtained has shown that, the sera (at 10-fold diluted concentration) from symptomatic and asymptomatic solubilised antigen immunised mice were able to specifically lyse the respective live parasites with an average percentage of 82% and 86% respectively. There were almost 50% crossreactivity observed between the three isolates of Blastocystis sp. ST3 from symptomatic and asymptomatic group proving high antigen diversity or rather low antigen specificity within the same group. However, there was only 17% cross-reactivity observed between the mice sera and parasitic cells of different groups (symptomatic vs asymptomatic isolates) suggesting high specificity between these two groups. We, for the first time have proven that through CDC analysis there were epitopes dissimilarities between Blastocystis sp. ST3 symptomatic and asymptomatic isolates which may allow the parasite to set up diverse immune modulations such as imbalanced Th1/Th2 responses in an infected host.

Matched MeSH terms: Antigens, Protozoan/immunology*

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