Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Recent Incidence of Paragonimus westermani Metacercariae in Freshwater Crayfish, Cambaroides similis, from Two Enzootic Sites in Jeollanam-do, Korea

An epidemiological study was performed to know the recent infection status of Paragonimus westermani metacercariae (PwMc) in freshwater crayfish, Cambaroides similis, from 2 streams in Jeollanam-do, Republic of Korea. Crayfish were collected from creeks in Bogil-do (Island), Wando-gun, and in a creek near Daeheung Temple in Haenam-gun. The infection rate of crayfish with PwMc in Bogil-do was 89.8%, and the metacercarial burden was 37 PwMc per the infected crayfish. Crayfish in a creek near Daeheung Temple were larger and twice heavier than those in Bogil-do. Of them, 96.5% were infected with PwMc. An average of 140 metacercariae was found in the infected crayfish, almost quadruple to those of Bogil-do. There was a strong correlation between the number of PwMc and body weight of the crayfish. These results suggest that P. westermani metacercariae are still prevalent in crayfish of the 2 regions in Jeollanam-do, Korea.

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.

Four Human Cases of Diphyllobothrium latum Infection

Diphyllobothrium latum infections in 4 young Korean men detected from 2008 to 2012 are presented. Three were diagnosed based on spontaneously discharged strobila of the adult worm in their feces, and 1 case was diagnosed by finding the worm at colonoscopy examination in a local clinic. The morphologic characteristics of the gravid proglottid and eggs were consistent with D. latum. All patients were treated with praziquantel 15 mg/kg, and follow-up stool examinations were done at 2 months after the medication. The main clinical complaints were intermittent gastrointestinal troubles such as indigestion, abdominal distension, and spontaneous discharge of tapeworm's segments in their feces. The most probable source of infection was the flesh of salmon or trout according to a patient's past history. These are the 45th to 48th recorded cases diagnosed by the adult worm in the Republic of Korea since 1971.

Electron Microscopy of the Separated Outer Tegument of the Sparganum and Its Antigenicity

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.

Experimental Murine Fascioliasis Derives Early Immune Suppression with Increased Levels of TGF-beta and IL-4

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19+ B cells was observed as early as week 1 post-infection while CD4+/CD8+ T cells were down-regulated. Accumulation of Mac1+ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-alpha mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1beta expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-beta were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-beta and IL-4 during the early stages of infection.

Expression of TNF-alpha and IL-1beta in Splenic Dendritic Cells and Their Serum Levels in Mouse Sparganosis

Sparganosis is a tissue invading helminthiasis infecting intermediate hosts, including humans. Strong immune responses are expected to occur in early phases of infection. Thus, we investigated cytokine expressions in splenic dendritic cells and in sera after experimental infection of mice. In splenic dendritic cells, TNF-alpha and IL-1beta expression peaked at week 1 and week 3 post-infection (PI), respectively, and also early phase (week 2 PI) depressed cytokine expression was noticed. Serum IL-1beta concentration increased significantly at week 2 PI and peaked at week 6 PI, and that of TNF-alpha peaked at week 6 PI. These results showed that pro-inflammatory cytokines, TNF-alpha and IL-1beta, are chronologically regulated in mouse sparganosis.

Pro-inflammatory Cytokine Expression of Spleen Dendritic Cells in Mouse Toxoplasmosis

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1alpha, IL-1beta, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8alpha+/CD11c+ splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.

Production of Polyclonal Antibodies against the Tegument of Sparganum (Plerocercoid of Spirometra mansoni) and Its Immunolocalization

In a previous study, the author developed a method for separation of the tegument of spargana (plerocercoids of Spirometra mansoni) from the parenchyme using urea. The present study, as a next step, was performed to evaluate which molecules are present in the outer tegument. Two major proteins, 180 and 200 kDa, are present in the tegument and we could make polyclonal antibodies against these molecules. Their immunolocalization was processed and the outermost layer of the spargana showed strong positive staining. Conclusively, we could confirm that the 180 and 200 kDa molecules might be tightly bound membrane proteins in the tegument of spargana.

Separation of the Syncytial Layer of Spargana using Urea

The tegument of tapeworms is known to be composed of an outer syncytial cytoplasm layer which includes microtriches and cytoplasmic organelles (= syncytial layer), and a parenchymatous cytoplasm layer that contains subtegumental cell nuclei (= subtegumental layer) and organelles. In the present study, separation of the syncytial layer of the sparganum, the plerocercoid stage of Spirometra mansoni, was tried using urea as the chemical reagent. Histological sections were prepared to visualize the status of separation after staining with hematoxylin and eosin. The results showed that the syncytial layer of the sparganum tegument which includes microtriches and cytoplasmic organelles were successfully separated from the parenchyma using 3 M urea.

Sympatric Distribution of Three Human Taenia Tapeworms Collected between 1935 and 2005 in Korea

Taeniasis has been known as one of the prevalent parasitic infections in Korea. Until recently, Taenia saginata had long been considered a dominant, and widely distributed species but epidemiological profiles of human Taenia species in Korea still remain unclear. In order to better understand distribution patterns of human Taenia tapeworms in Korea, partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were determined, along with morphological examinations, on 68 Taenia specimens obtained from university museum collections deposited since 1935. Genomic DNA was extracted from formalin-preserved specimens. Phylogenetic relationships among the genotypes (cox1 haplotype) detected in this study were inferred using the neighbor-joining method as a tree building method. Morphological and genetic analyses identified 3 specimens as T. solium, 51 specimens as T. asiatica, and 14 specimens as T. saginata. Our results indicate that all 3 Taenia tapeworms are sympatrically distributed in Korea with T. asiatica dominating over T. saginata and T. solium.

Partial Purification and Characterization of a Cysteine Protease Inhibitor from the Plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.

Changes of Serum Adiponectin Levels in Murine Experimental Sparganosis

The weight gain phenomenon associated with sparganosis has been well documented and was first recognized n the 1960s. Many studies have been conducted regarding the plerocercoid growth factor in the larva of Spirometra mansoni. n the present study, we hypothesized that the weight gain may be affected by the adipocyte secreted hormones, i.e., diponectin, which is secreted from the adipose tissues in case of tissue migrating parasitic infections. Specifically, we ttempted to ascertain whether the serum levels of adiponectin change in murine sparganosis. However, serum adiponectin levels assayed by ELISA evidenced no significant changes after an experimental infection (P > 0.05). Finally, the weight gain phenomenon in mouse sparganosis is not associated with changes in adiponectin levels, and further investigations involving parasitic infection-induced weight gain remain necessary.

Feminization and reduction of testicular weight in mouse sparganosis

After infection of male mice with the plerocercoids (spargana) of Spirometra mansoni, serum levels of estrogen and testicular weight were analyzed by enzyme-linked immunosorbent assay (ELISA) and weighing machine, respectively. The serum level of estrogen increased progressively in infected mice compared with normal controls, whereas the testicular weight of infected mice decreased significantly (P < 0.05). These results suggest that certain substances from spargana change the steroid hormone metabolisms in the host by unknown pathways, and chronic infection may contribute to change of the function of steroid hormone target organ, i.e., testis, in male mice.

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.

Enzymatic N-glycan analysis of 31 kDa molecule in plerocercoid of Spirometra mansoni (sparganum) and its antigenicity after chemical oxidation

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

Modification of carbohydrate compositions of 31/36 kDa proteins of plerocercoids (sparganum) of Spirometra mansoni grown in different intermediate hosts

We purified specific 31/36 kDa antigenic molecules from sparganum in different intermediate hosts (snakes and mice) and analyzed their monosaccharide compositions. Compositional analysis showed that glucose and mannose concentrations were 2-3 fold higher in the 31/36 kDa molecule purified from snakes than those from mice. This result implies that antigenic glycoproteins of sparganum from snakes might be modified in mammalian sparganosis with respect to their carbohydrate composition.

Immunolocalization of the 150 kDa protein in cyst fluid of Taenia solium metacestodes

The 150 kDa protein of cyst fluid (CF) of Taenia solium metacestodes was purified by ammonium sulfate fractionation and Superose 6 HR gel filtration chromatography. The purified protein consisted of three subunits (15, 10 and 7 kDa proteins), which were analyzed with the use of a 7.5-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunofluorescence study was carried out by using immunize specific polyclonal antibody. Positive reactions were noticed at bladder walls, calcareous corpuscles, granules of cyst fluid and some host tissue surrounding the bladder wall of the metacestodes. These results suggest that the 150 kDa protein was secreted into host tissues, inducing immune responses in the host, and it may play important roles in the cellular physiology of the parasites.

Immunoblot findings of calcareous corpuscles binding proteins in cyst fluid of Taenia solium metacestodes

After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.