107 results on '"Grams, Rudi"'
Search Results
2. Current prevalence and geographic distribution of helminth infections in the parasitic endemic areas of rural Northeastern Thailand
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Martviset, Pongsakorn, Phadungsil, Wansika, Na-Bangchang, Kesara, Sungkhabut, Wiwat, Panupornpong, Tanutchamon, Prathaphan, Parisa, Torungkitmangmi, Nattaya, Chaimon, Salisa, Wangboon, Chompunoot, Jamklang, Mantana, Chumkiew, Sirilak, Watthanasiri, Pichanee, Geadkaew-Krenc, Amornrat, Grams, Rudi, Mungthin, Mathirut, and Chantree, Pathanin
- Published
- 2023
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3. Coproprevalence, seroprevalence, and geographic distribution of Fasciola spp. infection in beef and dairy cattle in Pak Chong highland, Nakhon-Ratchasima Province, Northeast Thailand
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Martviset, Pongsakorn, Geadkaew-Krenc, Amornrat, Piyatadsananon, Pantip, Jirojwong, Ruttiroj, Chantree, Pathanin, Phadungsil, Wansika, Wangboon, Chompunoot, Jamklang, Mantana, Chumkiew, Sirilak, Poomkhokrak, Rawipreeya, Taylor, Aree, Kosa, Nanthawat, and Grams, Rudi
- Published
- 2024
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4. Investigation of a Serine Protease Inhibitor Active in the Infectious Stage of the Human Liver Fluke Opisthorchis viverrini.
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Salang, Rosnanee, Phadungsil, Wansika, Geadkaew-Krenc, Amornrat, and Grams, Rudi
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OPISTHORCHIS viverrini ,SERINE proteinases ,LIVER flukes ,REVERSE transcriptase ,PROTEASE inhibitors - Abstract
Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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5. Improvement of a PCR-based method for the detection of Opisthorchis viverrini eggs in human stool samples by targeting internal transcribed spacer-2 (ITS-2), cytochrome oxidase subunit 1 (cox1), and cytochrome b (cyb)
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Pumpa, Supaporn, Phadungsil, Wansika, Grams, Rudi, Martviset, Pongsakorn, Ruang-Areerate, Toon, Mungthin, Mathirut, and Geadkaew-Krenc, Amornrat
- Published
- 2021
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6. Cystatins from the Human Liver Fluke Opisthorchis viverrini : Molecular Characterization and Functional Analysis.
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Geadkaew-Krenc, Amornrat, Grams, Rudi, Siricoon, Sinee, Kosa, Nanthawat, Krenc, Dawid, Phadungsil, Wansika, and Martviset, Pongsakorn
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OPISTHORCHIS viverrini ,CYSTATINS ,LIVER flukes ,CYSTEINE proteinase inhibitors ,CHOLANGIOCARCINOMA ,SERINE proteinases - Abstract
A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1–6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3–6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3–9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated. [ABSTRACT FROM AUTHOR]
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- 2023
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7. Proteomic profile of Bithynia siamensis goniomphalos snails upon infection with the carcinogenic liver fluke Opisthorchis viverrini
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Prasopdee, Sattrachai, Tesana, Smarn, Cantacessi, Cinzia, Laha, Thewarach, Mulvenna, Jason, Grams, Rudi, Loukas, Alex, and Sotillo, Javier
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- 2015
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8. Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties
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Janvilisri, Tavan, Likitponrak, Wichai, Chunchob, Supatra, Grams, Rudi, and Vichasri-Grams, Suksiri
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- 2007
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9. Improved Allele-Specific PCR Technique for Kidd Blood Group Genotyping
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Intharanut, Kamphon, Grams, Rudi, Bejrachandra, Sasitorn, Sriwanitchrak, Pramote, and Nathalang, Oytip
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- 2013
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10. Opisthorchis viverrini: Identification of a glycine–tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis
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Ruangsittichai, Jiraporn, Viyanant, Vithoon, Vichasri-Grams, Suksiri, Sobhon, Prasert, Tesana, Smarn, Upatham, Edward Suchart, Hofmann, Annemarie, Korge, Günter, and Grams, Rudi
- Published
- 2006
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11. Comparative analysis of two fatty acid binding proteins from Fasciola gigantica
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CHUNCHOB, SUPATRA, GRAMS, RUDI, VIYANANT, VITHOON, SMOOKER, PETER M., and VICHASRI-GRAMS, SUKSIRI
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- 2010
12. Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs.
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Phadungsil, Wansika, Pumpa, Supaporn, Sirisabhabhorn, Kridsada, Geadkaew-Krenc, Amornrat, Grams, Rudi, Mungthin, Mathirut, Ruang-Areerate, Toon, Adisakwattana, Poom, Labbunruang, Nipawan, and Martviset, Pongsakorn
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OPISTHORCHIS viverrini ,NADH dehydrogenase ,CHOLANGIOCARCINOMA ,MIXED infections ,EGGS ,HELMINTHS ,TRYPANOSOMA cruzi - Abstract
Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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13. Morphology and Mitochondrial Genome of Fischoederius sp. 1 in Thailand.
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Watthanasiri, Pichanee, Geadkaew-Krenc, Amornrat, and Grams, Rudi
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MITOCHONDRIA ,MORPHOLOGY ,INVERTED repeats (Genetics) ,GENOMES ,SPECIES - Abstract
A rumen fluke Fischoederius elongatus is assigned to the type species of genus Fischoederius, family Gastrothylacidae. However, the mitochondrial sequences recently published are thought to be of inconsistent species, suggesting that several morphologically similar but genetically distinct species might be classified as Fischoederius elongatus. Thus, mentions of F. elongatus from South, Southeast, and East Asia might unintentionally refer to different species. The present work describes morphology and a full mitochondrial genome sequence of one of these species. The fluke specimens were collected from 2 infected cattle in Thailand. An interesting finding was the presence of a second tRNA-Asp gene next to a partial ND1 gene. It is suggested that these duplicated sequences are the remnants of non-reciprocal recombination events caused by inverted repeats located between ND2 and ND1 mitochondrial genes. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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14. Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin.
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Phadungsi, Wansika and Grams, Rudi
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FASCIOLA ,CRASSOSTREA ,ABO blood group system ,PACIFIC oysters ,LECTINS ,ANOPHELES gambiae ,AGGLUTINATION - Abstract
The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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15. Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica
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Chaithirayanon, Kulathida, Wanichanon, Chaitip, Vichasri-Grams, Suksiri, Ardseungneon, Pissanee, Grams, Rudi, Viyanant, Vithoon, Upatham, Edward Suchart, and Sobhon, Prasert
- Published
- 2002
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16. Evaluation of Rhophilin Associated Tail Protein (ROPN1L) in the Human Liver Fluke Opisthorchis viverrini for Diagnostic Approach.
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Geadkaew-Krenc, Amornrat, Grams, Rudi, Phadungsil, Wansika, Chaibangyang, Wanlapa, Kosa, Nanthawat, Adisakwattana, Poom, and Dekumyoy, Paron
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OPISTHORCHIS viverrini ,LIVER flukes ,TAILS ,PARASITIC diseases ,CYSTICERCOSIS ,PROTEINS ,CLONORCHIS sinensis - Abstract
Tegumental and excretory-secretory proteins are reported as diagnostic antigens for human opisthorchiasis. Rhophilin associated tail protein1-like (OvROPN1L) protein of Opisthorchis viverrini sperm tail showed potential as a diagnostic antigen. The OvROPN1L recombinant fragments were assayed for diagnostic antigenicity for human opisthorchiasis using indirect ELISA. The strongest antigenic region was a N-terminus peptide of M1 - P56. One synthetic peptide (P1, L3-Q13) of this region showed the highest antigenicity to opisthorchiasis. Sera from other parasitic infections including Strongyloides stercoralis, hookworm, Taenia spp, minute intestinal flukes, Paragonimus spp showed lower reactivity to P1. Peptide P1 is located in the disordered N-terminus of ROPN1L supporting its suitability as linear epitope. In the Platyhelminthes the N-terminal sequence of ROPN1L is diverging with taxonomic distance further suggesting that peptide P1 has potential as diagnostic tool in the genus Opisthorchis/Clonorchis. It should be further evaluated in combination with peptides derived from other O. viverrini antigens to increase its diagnostic power. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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17. FASCIOLA GIGANTICA CATHEPSIN B8, AN ISOFORM PRESENT FROM METACERCARIAL TO MATURE STAGE.
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Lwin, Thwet Oo, Geadkaew-Krenc, Amornrat, Siricoon, Sinee, and Grams, Rudi
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- 2020
18. Data set from the proteomic analysis of Bithynia siamensis goniomphalos snails upon infection with the carcinogenic liver fluke Opisthorchis viverrini
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Prasopdee, Sattrachai, Tesana, Smarn, Cantacessi, Cinzia, Laha, Thewarach, Mulvenna, Jason, Grams, Rudi, Loukas, Alex, and Sotillo, Javier
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- 2015
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19. Comparative Characterization of Four Calcium-Binding EF and Proteins from Opisthorchis viverrini.
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Emmanoch, Palida, Kosa, Nanthawat, Vichasri-Grams, Suksiri, Tesana, Smarn, Grams, Rudi, and Geadkaew-Krenc, Amornrat
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OPISTHORCHIS viverrini ,DYNEIN ,WESTERN immunoblotting ,PLATYHELMINTHES ,CHOLANGIOCARCINOMA - Abstract
Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2-42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21-23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca
, Mg , Zn , and Cu . All OvCaBPs showed mobility shifts with Ca and Zn . OvCaBP1 showed also positive results with Mg and Cu . As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis. [ABSTRACT FROM AUTHOR] - Published
- 2018
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20. Molecular and Biochemical Characterization of Opisthorchis viverrini Calreticulin.
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Chaibangyang, Wanlapa, Geadkaew-Krenc, Amornrat, Vichasri-Grams, Suksiri, Tesana, Smarn, and Grams, Rudi
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OPISTHORCHIS viverrini ,CALRETICULIN ,APOPTOTIC bodies ,ACETYLTRANSFERASES ,CITRATE synthase - Abstract
Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P- and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehli's gland, prostate gland and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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21. Opisthorchis viverrini: Analysis of the sperm-specific rhophilin associated tail protein 1-like.
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Rattanachan, Sitthichon, Grams, Rudi, Tesana, Smarn, Smooker, Peter M., and Grams, Suksiri Vichasri
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OPISTHORCHIS viverrini , *CARRIER proteins , *SPERMATOZOA , *IMMUNOHISTOCHEMISTRY , *REVERSE transcriptase polymerase chain reaction , *IMMUNOELECTRON microscopy - Abstract
Concurrent deficiency of rhophilin associated tail protein (ROPN1) and ROPN1-like (ROPN1L) in mice causes structural abnormalities and immotility of sperm and thereby infertility. In the present research, ROPN1L of the human liver fluke Opisthorchis viverrini was molecularly characterized and showed unexpected potential as a diagnostic tool. ROPN1L transcripts were detected in 2-week-old juveniles by RT-PCR. Immunohistochemical analysis of the adult worm localized the protein in testis lobes, seminal vesicle and receptacle and immunoelectron microscopic analysis revealed its location on the tail of spermatozoa. Interestingly, sera of experimentally infected hamsters and sera of individuals suffering from opisthorchiasis showed reactivity to recombinant OvROPN1L (rOvROPN1L). The protein shows modest conservation to the human homolog at 47.2% sequence identity and a mouse anti-rOvROPN1L antiserum was not reactive with sperm protein extracts from hamsters, mice and rats. Unsurprisingly, conservation is higher in trematodes, e.g. 78.4% and 71.2% identity to Fasciola gigantica and Schistosoma haematobium , respectively and evaluation of diagnostic specificity is required using sera of individuals suffering from different trematodiases in Thailand. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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22. Analysis of a calcium-binding EF-hand protein family in Fasciola gigantica
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Subpipattana, Pornpimol, Grams, Rudi, and Vichasri-Grams, Suksiri
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CALCIUM-binding proteins , *FASCIOLA , *DATA analysis , *PLATYHELMINTHES , *SCHISTOSOMA , *LIVER flukes , *LABORATORY rats - Abstract
Abstract: Transcriptome data supports the notion of a Platyhelminthes-specific protein family that is characterized by combination of two N-terminal EF-hands and a C-terminal dynein light chain-like domain. Family members in schistosomes induce an IgE response that has been connected with resistance to reinfection in schistosomiasis and is considered as a marker of protection. In the present study, we have compared three homologs of the liver fluke Fasciola gigantica for their immunological properties in mouse. Antisera raised against the recombinant proteins detected the native proteins in tegumental type tissues and epithelial linings of excretory system and intestinal tract. The recombinant EF-hand domains induced strong IgG and IgE responses in immunised mice while only weak to moderate responses were observed against the complete recombinant proteins and their DLC-like domains. Parasite crude worm and tegumental extract antisera reacted predominantly with one isoform and its EF-hand domain. Sera of F. gigantica infected mice did not react with the recombinant proteins. The RNA products of the three genes were detected from the metacercarial up to the adult stage. These observations indicate that the investigated EF-hand proteins are not at the frontier of humoral host/parasite interaction in acute fascioliasis gigantica in mouse but are acting as intracellular proteins in tissues that interface with the parasite’s environment or tubular tracts. [Copyright &y& Elsevier]
- Published
- 2012
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23. Confutation of the existence of sequence-conserved cytochrome P450 enzymes in Plasmodium falciparum
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Wisedpanichkij, Raewadee, Grams, Rudi, Chaijaroenkul, Wanna, and Na-Bangchang, Kesara
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CYTOCHROME P-450 , *PLASMODIUM falciparum , *BIOINFORMATICS , *NUCLEOTIDE sequence , *RNA , *ANTISENSE DNA , *NUCLEIC acid hybridization - Abstract
Abstract: The aim of the present study was to find evidence for a homologous protein of the mammalian cytochrome P450 family member CYP2B1/B2 in Plasmodium falciparum at the nucleic acid level. Prior research had demonstrated enzyme activity in the parasite comparable to mammalian CYP1A, 2A, 2B and 2E enzymes and presence of CYP enzymes by spectrophotometric and electrophoretic analyses. In recent years, the transcriptome/proteome data of P. falciparum and other Plasmodium spp. have been published and we performed an in silico analysis to identify putative cytochrome P450 family members in the parasite. This analysis failed to identify homologs to CYP1A, 2A, 2B and 2E enzymes in Plasmodium. A prior study had also claimed the presence of a conserved CYP2B1/B2 gene in the parasite by using Northern analysis with a rat CYP2B1/B2 probe. We have repeated this analysis by cloning a rat CYP2B1/B2 cDNA and using it as a hybridization probe against total RNA extracted from P. falciparum K1 and 3D7 clones but did not obtain positive results. This is consistent with the transcriptome/proteome sequence data and suggests that the genus Plasmodium contains either only highly diverged CYP proteins which are not easily identified by their primary sequence or that they have been functionally replaced by other enzymes. It is suggested that further studies are performed that allow isolation and identification of such proteins through their functional activities. [Copyright &y& Elsevier]
- Published
- 2011
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24. The saposin-like proteins 1, 2, and 3 of Fasciola gigantica
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Grams, Rudi, Adisakwattana, Poom, Ritthisunthorn, Nonglucksanawan, Eursitthichai, Veerachai, Vichasri-Grams, Suksiri, and Viyanant, Vithoon
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FASCIOLA , *PROTEINS , *ERYTHROCYTES , *OLIGONUCLEOTIDES - Abstract
Abstract: The SAP genes of Fasciola encode proteins belonging to the saposin-like protein family. The saposin signature, a compact domain of mainly α-helical character, contains six conserved cysteine residues and has been implicated in membrane-binding, pore formation, and subsequent cell lysis in several family members. Recombinant SAP-2 of F. hepatica has been shown to induce lysis of human erythrocytes and peripheral blood mononuclear cells. This suggests that the SAPs are involved in the nutrition of Fasciola as the released content of lysed host cells is available for further enzymatic processing and uptake by the parasite. In the present study a new SAP-3 cDNA was obtained in an immunoscreen of an adult stage F. gigantica cDNA library with an antiserum against the parasite''s excretion/secretion antigens. SAP-1 and SAP-2 cDNAs were isolated from F. gigantica cDNA libraries using oligonucleotide primers specific to the SAP-1 and SAP-2 DNA sequences from F. hepatica. Transcripts of the three SAPs are present from the metacercarial to the adult stage and are located to the gut epithelium. In immatures SAP-1 RNA is the predominant product whereas in adults SAP-2 and -3 are the more abundant products. Polyclonal anti-SAP-1 and SAP-2 antisera confirmed the tissue-specificity and revealed the subcellular localization of SAPs in large granules concentrated in the apical part of the gut epithelial cells of the parasite. Interestingly, evolutionary conservation of the Fasciola SAP sequences among other trematodes is low at 20–30% sequence identity comparable to the Entamoeba amoebapore sequences. [Copyright &y& Elsevier]
- Published
- 2006
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25. Molecular Cloning and Characterization of a Glutathione S-Transferase Encoding Gene from Opisthorchis viverrini.
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Eursitthichai, Veerachai, Viyanant, Vithoon, Vichasri-Grams, Suksiri, Sobhon, Prasert, Tesana, Smarn, Upatham, Suchart Edward, Hofmann, Annemarie, Korge, Günter, and Grams, Rudi
- Published
- 2004
26. Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica
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Meemon, Krai, Grams, Rudi, Vichasri-Grams, Suksiri, Hofmann, Annemarie, Korge, Günter, Viyanant, Vithoon, Upatham, Edward Suchart, Habe, Shigehisa, and Sobhon, Prasert
- Subjects
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MOLECULAR cloning , *GENE expression , *FASCIOLA , *PROTEINS - Abstract
The transcriptional products of Fasciola gigantica genes encoding cathepsin B proteases were cloned from adult, newly excysted juvenile (NEJ), and metacercarial stages. The obtained cDNAs were named FG cat-B1, FG cat-B2, and FG cat-B3. The deduced amino acid sequences of the encoded proteases have identities ranging from 64 to 79%. Sequence comparison with homologous proteins showed that all functional important residues formerly described for cathepsin B are conserved. Southern analysis confirmed the presence of a family of related cathepsin B genes in the genome of F. gigantica. Northern analysis revealed a common transcript size of 1400 nucleotides with abundant cathepsin B transcripts detected in metacercarial and NEJ stages. Cathepsin B transcripts were located by RNA in situ hybridization in the caecal epithelial cells, in cells underlining the proximal part of the digestive tract, and in the tegumental cells underlining the surface tegument. Furthermore, transcripts were detected in the tissues of the reproductive system including cells of prostate, Mehlis, and vitelline glands, testis, and eggs. Stage-specific gene expression was investigated by RT-PCR using gene-specific primers and hybridization with a labeled cathepsin B probe. FG cat-B1 transcripts were detected in all stages, whereas FG cat-B2 and FG cat-B3 transcripts were expressed in metacercariae, NEJ, and juvenile parasites only. The switching off of the cat-B2 and cat-B3 genes during the maturation of the parasites implicates that these enzymes may be involved in digesting host tissues during penetration and migration to the liver, whereas cat-B1 present in all stages may perform general digestive function. [Copyright &y& Elsevier]
- Published
- 2004
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27. Production and Characterization of a Monoclonal Antibody against Recombinant Glutathione S-Transferase (GST) of Fasciola gigantica.
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Khawsuk, Witoon, Soonklang, Nantawan, Grams, Rudi, Vichasri-Grams, Suksiri, Wanichanon, Chaitip, Meepool, Ardool, Chaithirayanon, Kulathida, Ardseungneon, Pissanee, Viyanant, Vithoon, Upathum, Suchart Edward, and Sobhon, Prasert
- Published
- 2002
28. A SINGLE PARATHYROID HORMONE RECEPTOR-LIKE MEMBER OF FAMILY B1 G-PROTEIN COUPLED RECEPTORS IN FASCIOLA GIGANTICA.
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Martviset, Pongsakorn and Grams, Rudi
- Published
- 2017
29. Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica
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Siricoon, Sinee, Grams, Suksiri Vichasri, and Grams, Rudi
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CYSTEINE proteinases , *CATHEPSIN B , *FASCIOLA , *ETIOLOGY of diseases , *ANTIBIOTICS in nutrition , *GENITALIA , *VACCINES - Abstract
Abstract: Cysteine proteases are important antigens in the liver fluke genus Fasciola, essential for infection, protection and nutrition. While their biochemistry, biological roles and application as vaccines have been thoroughly studied there is a lack of data concerning their regulation. In the present study we have continued our investigation of cysteine protease inhibitors in Fasciola gigantica and demonstrate, in comparison with FgStefin-1 and human cystatin C, that a second type 1 cystatin of the parasite, FgStefin-2, has been evolutionary adapted to block cathepsin B. The protein, which unusually for a type 1 cystatin carries a signal peptide, is expressed from the metacercarial to adult stage and located in the epithelial cells of the intestinal tract in all stages and in the prostate gland cells in adults. Both cell types may contribute to the released FgStefin-2 observed in the ES product of the parasite. Distinct isoforms of cathepsin B are essential for host tissue penetration during the early infection process and FgStefin-2 may act as key regulator, required to protect the minute juvenile from autoproteolysis. Expression in the prostate gland in the adult stage suggests an additional regulative role of cysteine protease activity in the reproductive system. [Copyright &y& Elsevier]
- Published
- 2012
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30. Classification of the parenchymal cells in Fasciola gigantica based on ultrastructure and their expression of fatty acid binding proteins (FABPs)
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Pankao, Viriya, Sirisriro, Anuchittada, Grams, Rudi, Vichasri-Grams, Suksiri, Meepool, Ardool, Kangwanrangsan, Niwat, Wanichanon, Chaitip, Ardseungneon, Pissanee, Viyanant, Vithoon, Upatham, Edward Suchart, and Sobhon, Prasert
- Subjects
- *
FASCIOLA , *CARRIER proteins , *PROTOPLASM , *MITOCHONDRIA - Abstract
Abstract: Parenchymal cells in adult Fasciola gigantica can be classified into three types based on their ultrastructural features and different quantities of fatty acid binding protein (FABP) being stored. Parenchymal cell type 1 (Pc1) has pale cytoplasm consisting largely of a loose network of fine fibers, and it contains few mitochondria but numerous glycogen particles. This cell type may be specialized in the storage and metabolism of glycogen and glucose. Parenchymal cell type 2 (Pc2) has similar cytoplasmic features as Pc1 but contains more numerous mitochondria, and high concentration of FABP as reflected by high density of immunostaining and immunogold labeling using specific monoclonal antibody (MoAb) to FABP as probe. Pc2 may, thus, specialize in the storage and metabolism of fatty acids and other lipids. Parenchymal cell type 3 (Pc3) has dense cytoplasm containing large amount of rough endoplasmic reticulum, Golgi complex and mitochondria, which is typical of a secretory cell. Furthermore, Pc3 has very little glycogen particles and is not stained by MoAb against FABP. It could, thus, be concerned with the synthesis of fibers, which form the scaffold of the parenchyma. [Copyright &y& Elsevier]
- Published
- 2006
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31. Evaluation of Opisthorchis viverrini calreticulin for potential host modulation.
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Chaibangyang, Wanlapa, Geadkaew-Krenc, Amornrat, Smooker, Peter M., Tesana, Smarn, and Grams, Rudi
- Subjects
- *
OPISTHORCHIS viverrini , *CALRETICULIN , *CALCIUM , *CARRIER proteins , *ENDOPLASMIC reticulum - Abstract
The multifunctional calreticulin (CALR) was identified as a major calcium-binding protein of the endoplasmic reticulum before being recognized as a chaperone in the same place. Only later were activities of calreticulin outside the endoplasmic reticulum described that for example affect cell proliferation and the innate immune system. In the present work we have investigated those extracellular activities of CALR from the cancerogenic human liver fluke Opisthorchis viverrini (OvCALR), as they might be important in host/parasite interaction. We first demonstrate that OvCALR is released from the parasite and stimulates a specific humoral immune response. Recombinant OvCALR is then shown to suppress proliferation of primary endothelial cells, their motility and sprouting activities. The potential of OvCALR to interfere with the complement system is established, firstly by demonstrating its direct binding to C1q and, secondly by suppression of hemolysis of sensitized red blood cells. These findings suggest that OvCALR is an important parasite antigen that could modulate diverse host functions and support parasite survival. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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32. Similarity of a 16.5 kDa tegumental protein of the human liver fluke Opisthorchis viverrini to nematode cytoplasmic motility protein.
- Author
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Labbunruang, Nipawan, Phadungsil, Wansika, Tesana, Smarn, Smooker, Peter M., and Grams, Rudi
- Subjects
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LIVER flukes , *CHOLANGIOCARCINOMA , *OPISTHORCHIS viverrini , *NEMATODES , *MICROBODIES , *ENZYME-linked immunosorbent assay , *DIAGNOSIS - Abstract
Opisthorchis viverrini is the causative agent of human opisthorchiasis in Thailand and long lasting infection with the parasite has been correlated with the development of cholangiocarcinoma. In this work we have molecularly characterized the first member of a protein family carrying two DM9 repeats in this parasite (OvDM9-1). InterPro and other protein family databases describe the DM9 repeat as a protein domain of unknown function that has been first noted in Drosophila melanogaster . Two paralogous proteins have been partially characterized in the genus Fasciola, Fasciola hepatica TP16.5, a novel tegumental antigen in human fascioliasis and, recently F. gigantica DM9-1, a parenchymal protein with structural similarity to nematode cytoplasmic motility protein (MFP2). In this study, we show further evidence that this family of trematode proteins is related to MFP2 in sequence and structure. Soluble recombinant OvDM9-1 was used for structural analyses and for production of specific antisera. The native protein was detected in soluble and insoluble crude worm extracts and in seemingly various oligomeric forms in the latter. The potential for oligomerization was supported by cross-linking experiments of recombinant OvDM9-1. Structure prediction suggested a β-rich secondary structure of the protein and this was supported by a circular dichroism analysis. Molecular modeling in Phyre2 identified both MFP2 domains as distant homologs of OvDM9-1. The protein was located in tegumental type tissue and the cecal epithelium in the mature parasite. Recombinant OvDM9-1 was used as target in indirect ELISA but sera from infected hamsters showed only marginal reactivity towards it. It is proposed that OvDM9-1 and other members of this protein family have a role in cellular transport through functions on the cytoskeleton. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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33. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.
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Phadungsil, Wansika, Smooker, Peter M., Vichasri-Grams, Suksiri, and Grams, Rudi
- Subjects
- *
FASCIOLA , *DROSOPHILA proteins , *PROTEOMICS , *TANDEM repeats , *INSECT parasites , *OLIGOMERIZATION - Abstract
Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60–75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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34. Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.
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Siricoon, Sinee, Vichasri Grams, Suksiri, Lertwongvisarn, Kittisak, Abdullohfakeeyah, Muntana, Smooker, Peter M., and Grams, Rudi
- Subjects
- *
FASCIOLA , *CATHEPSIN B , *CYSTEINE proteinase inhibitors , *CYSTEINE proteinases , *GENE expression , *ENZYME-linked immunosorbent assay - Abstract
Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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35. Bi-functionality of Opisthorchis viverrini aquaporins.
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Geadkaew, Amornrat, von Bülow, Julia, Beitz, Eric, Tesana, Smarn, Vichasri Grams, Suksiri, and Grams, Rudi
- Subjects
- *
OPISTHORCHIS viverrini , *AQUAPORINS , *ORGANISMS , *REGULATION of body fluids , *MEMBRANE proteins , *DRUG therapy , *TARGETED drug delivery - Abstract
Aquaporins (AQP) are essential mediators of water regulation in all living organisms and members of the major intrinsic protein (MIP) superfamily of integral membrane proteins. They are potential vehicles or targets for chemotherapy, e.g. in Trypanosoma brucei melarsoprol and pentamidine uptake is facilitated by TbAQP-2. Transcriptome data suggests that there are at least three active aquaporins in the human liver fluke, Opisthorchis viverrini , OvAQP-1, 2 and 3, and crude RNA silencing of OvAQP - 1 and 2 has recently been shown to affect parasite swelling in destilled water. In the present work we demonstrate that OvAQP-3 is a major water-conducting channel of the parasite, that it can be detected from the newly excysted juvenile to the adult stage and that it is present in major tissues of the parasite. Furthermore, a comparative functional characterization of the three parasite AQPs was performed by using Xenopus oocyte swelling and yeast phenotypic assays. OvAQP-1, OvAQP-2, and OvAQP-3 were found to conduct water and glycerol while only the latter two were also able to conduct urea. In addition, all OvAQPs were found to transport ammonia and methylamine. Our findings demonstrate that the sequence-based classification into orthodox aquaporins and glycerol-conducting aquaglyceroporins is not functionally conserved in the parasite and implicate a broder range of functions for these channels. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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36. Temperature dependence of Opisthorchis viverrini infection in first intermediate host snail, Bithynia siamensis goniomphalos.
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Prasopdee, Sattrachai, Kulsantiwong, Jutharat, Piratae, Supawadee, Khampoosa, Panita, Thammasiri, Chalida, Suwannatrai, Apiporn, Laha, Thewarach, Grams, Rudi, Loukas, Alex, and Tesana, Smarn
- Subjects
- *
OPISTHORCHIS viverrini , *PARASITIC diseases , *SNAILS , *COLD-blooded animals , *METABOLISM , *POLYMERASE chain reaction , *BITHYNIA (Mollusks) , *TEMPERATURE effect , *DISEASES - Abstract
Determining of the success of a parasite's infectiveness in its snail host clearly depends on environmental conditions. Temperature, one of the most influential factors impinging on metabolism of cold-blooded animals, is believed to be an important factor in parasitic infection in snails. In order to elucidate the influence of temperature, sex and size of snails on infectivity of Opisthorchis viverrini to its first intermediate host, Bithynia siamensis goniomphalos , 960 snails were divided into 2 groups by sex. Each group was subdivided by their size into small and medium sub-groups. Each snail was fed with embryonated uterine-eggs of O. viverrini at different temperatures (16–37 °C, 3 °C intervals). Dissections were carried out 1, 7, 14, 28 and 56 days thereafter and detection of O. viverrini infection was undertaken by PCR using specific primers. Infection was strongly temperature-dependent, as temperature increases of 1 °C resulted in increased odds of infection 5.4% ( P < 0.01). A temperature of 34 °C gave the highest rate of infection of 44.14%. We also found that the odds of infection in small sized snails was 39.8% higher relative to medium sized snails ( P < 0.05). Relative to day 1, the decrease in the odds of infection was detected when the day post infection was longer ( P < 0.01). Proportion of infection in female was not different to male significantly. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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37. A 170 kDa multi-domain cystatin of Fasciola gigantica is active in the male reproductive system.
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Geadkaew, Amornrat, Kosa, Nanthawat, Siricoon, Sinee, Grams, Suksiri Vichasri, and Grams, Rudi
- Subjects
- *
CYSTATINS , *FASCIOLA , *MALE reproductive organs , *CYSTEINE proteinases , *MOLECULAR weights , *PROPROTEIN convertases , *IMMUNOHISTOCHEMISTRY - Abstract
Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170 kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often <20% and at only 26.8% between the highest matching domains 6 and 10. Several domains contain degenerated QVVAG core motifs and/or lack other important residues of active type 2 cystatins. Domain-specific antisera detected multiple forms of FgMDC ranging from <10 to >120 kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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38. Adult and juvenile Fasciola cathepsin L proteases: Different enzymes for different roles
- Author
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Norbury, Luke J., Beckham, Simone, Pike, Robert N., Grams, Rudi, Spithill, Terry W., Fecondo, John V., and Smooker, Peter M.
- Subjects
- *
FASCIOLA , *PROTEOLYTIC enzymes , *ACETIC acid , *EXCYSTMENT (Dormancy) , *COUMARINS , *FUNCTIONAL analysis , *BIOCHEMISTRY - Abstract
Abstract: Cathepsin proteases are promising vaccine or drug targets for prophylaxis or therapy against Fasciola parasites which express cathepsin L and B proteases during their development. These proteases are believed to be involved in important functions for the parasite, including excystment, migration, feeding and host immune evasion. Several cathepsin L transcripts, including FhCatL5, have been isolated from adult Fasciola, while certain cathepsin L proteases, including FgCatL1G, have only been identified in the juvenile forms of the parasite. In this study, Fasciola hepatica cathepsin FhCatL5 and F. gigantica FgCatL1G were expressed in yeast and their biochemical properties characterised and compared. The pH profiles of activity and stability of the two recombinant cathepsins was shown to differ, differences that are likely to be functionally important and reflect the environments into which the cathepsins are expressed in vivo. Biochemical analysis indicates that FgCatL1G can cleave substrates with proline residues at P2, a characteristic previously described for the adult cathepsin FhCatL2. FgCatL1G and FhCatL5 show differences in their host substrate digestion patterns, with different substrates cleaved at varying efficiencies. Functional analysis of a recombinant FhCatL5 L69W variant indicates that the residue at position 69 is important for the S2 subsite architecture and can influence substrate specificity. [Copyright &y& Elsevier]
- Published
- 2011
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39. Functional analysis of novel aquaporins from Fasciola gigantica
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Geadkaew, Amornrat, von Bülow, Julia, Beitz, Eric, Grams, Suksiri Vichasri, Viyanant, Vithoon, and Grams, Rudi
- Subjects
- *
AQUAPORINS , *FASCIOLA , *FASCIOLIASIS , *LIVER flukes , *RUMINANTS , *BIOLOGICAL membranes , *IMMUNOHISTOCHEMISTRY , *PLATYHELMINTHES , *OVARIES - Abstract
Abstract: Fascioliasis, caused by liver flukes of the genus Fasciola, is an important disease of ruminants. In order to identify a potential new drug target we have studied aquaporin (AQP) in Fasciola gigantica. AQPs facilitate the transport of water, glycerol and other small solutes across biological membranes. The structure, function, and pathology of AQPs have been extensively studied in mammals but data for AQPs from trematodes is still limited. In the present study, we have functionally characterized two closely related AQP isoforms, FgAQP-1 and FgAQP-2, from the trematode F. gigantica. Immunohistochemical analysis located the FgAQPs in the tegumental cells, their processes and the tegument itself. In addition, they were present in the epithelial linings of testes and ovary. Expression in Xenopus oocytes of these FgAQPs increased osmotic water permeability 3–4-fold but failed to increase glycerol and urea permeability. AQPs have two highly conserved NPA motifs that are important for the function of the channel pore. In FgAQP-1 and FgAQP-2 the first NPA motif is changed to TAA. Substitution of Thr with Asn in the TAA motif of FgAQP-1 increased its water permeability twofold but did not affect urea and glycerol impermeability while the substitution at the pore mouth of Cys204 by Tyr caused loss of water permeability. In addition, the FgAQPs did not increase methylamine and ammonia permeability after expression in yeast. In comparison to rat AQP-1 the described FgAQPs showed low water permeability and further in vivo analyses are necessary to determine their contribution to osmoregulation in Fasciola. [Copyright &y& Elsevier]
- Published
- 2011
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40. Rapid identification of lymnaeid snails and their infection with Fasciola gigantica in Thailand
- Author
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Kaset, Chollanot, Eursitthichai, Veerachai, Vichasri-Grams, Suksiri, Viyanant, Vithoon, and Grams, Rudi
- Subjects
- *
LYMNAEIDAE , *FASCIOLA , *PARASITIC diseases , *NUCLEOTIDE sequence , *VETERINARY parasitology , *FASCIOLIASIS , *MONOCLONAL antibodies - Abstract
Abstract: Freshwater snails of the family Lymnaeidae are the intermediate hosts of the liver fluke Fasciola worldwide. While distinct species have been identified at the molecular level in other parts of the world such data have not been published for Thailand. In this study we collected Lymnaeidae from different localities across Thailand and analyzed their 16S rDNA sequences as a molecular signature for classification. In addition to the ubiquitous Radix rubiginosa, we have confirmed the presence of Austropeplea viridis and Radix swinhoei, for the latter of which the ribosomal rDNA sequences are reported for the first time, in North-Thailand. Based on the obtained 16S rDNA data three primer pairs were designed that allowed rapid identification of these snail species by PCR. To determine their infection status, PCR primers for F. gigantica cathepsin L were used in parallel with the snail 16S rDNA species-specific primers in multiplex PCR analyses. Western blot analysis of total snail protein with a monoclonal anti-F. gigantica cathepsin L antibody confirmed positive cathepsin L PCR results. The developed diagnostic PCR will be of use in risk assessment for transmission of fascioliasis in Thailand. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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- View/download PDF
41. Opisthorchis viverrini: Evaluation of 28kDa glutathione S-transferase as diagnostic tool in human opisthorchiasis
- Author
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Eursitthichai, Veerachai, Viyanant, Vithoon, Tesana, Smarn, Sithithaworn, Paiboon, Kosa, Nanthawat, and Grams, Rudi
- Subjects
- *
GLUTATHIONE transferase , *OPISTHORCHIASIS , *PROTOZOAN diseases , *OPISTHORCHIIDA , *IMMUNODIAGNOSIS , *PARASITE antigens , *DIAGNOSIS - Abstract
Abstract: The liver fluke Opisthorchis viverrini is the agent of human opisthorchiasis in Thailand with a high prevalence observed in the rural population of north and northeastern regions of the country. A focus of research has therefore been the development of diagnostic tools to indicate infection by this parasite. In the present study, a 28kDa glutathione S-transferase of O. viverrini (OV28GST), which is found in the excretion/secretion product of the parasite, was evaluated for its application in diagnosis of human opisthorchiasis. Bacterially expressed and functionally active rOV28GST was used in immunoblots and indirect ELISA to detect anti-OV28GST antibody in sera of infected individuals. Crude whole worm extract, sera of uninfected individuals and a rabbit anti-rOV28GST antiserum were used as controls in the assays while positivity for parasite DNA by PCR and egg count in faeces were used as primary indicators of infection. The results showed weak or absent reactivity of the infected sera to immunoblotted rOV28GST and no significant difference in absorbance values when compared to uninfected sera in ELISA. In addition, a glutathione capture ELISA which was performed to test for circulating OV28GST in human and hamster sera showed negative results. In conclusion, OV28GST is not applicable as a diagnostic tool in established infections due to low specific antibody titre and abundance as circulating antigen. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF
42. Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product
- Author
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Tarasuk, Mayuri, Vichasri Grams, Suksiri, Viyanant, Vithoon, and Grams, Rudi
- Subjects
- *
NUCLEIC acids , *BIOMOLECULES , *GENETIC transformation , *NUCLEOTIDES - Abstract
Abstract: In the present study we describe type 1 cystatin, a cysteine protease inhibitor, as a major released antigen of the tropical liver fluke Fasciola gigantica (FgStefin-1). Immunohistochemical analysis showed that FgStefin-1 is abundant in (a) tissue of tegumental type, including oral and ventral sucker, pharynx, genital atrium, metraterm, cirrus and (b) the intestinal epithelium. Faint staining was observed in the epithelia of ovary and proximal uterus. Immunoblots showed the presence of FgStefin-1 in the parasite''s excretion/secretion (ES) product and immunodepletion demonstrated that FgStefin-1 herein is partially complexed with cathepsin L. Furthermore, quantitation of FgStefin-1 in comparison to cathepsin L in ES product and crude worm extract of adults supports a major external function of FgStefin-1 with an estimated 50% being released in at least equimolar amounts to cathepsin L. Sera of an experimentally infected rabbit reacted with recombinant FgStefin-1 starting 8 weeks postinfection. Activity analyses of recombinant FgStefin-1 showed nanomolar inhibition constants for mammalian cathepsin B, L, and S cysteine proteases and released cysteine proteases of the parasite. The protein is active over a wide pH range and is heat stable. Our results suggest protective functions of FgStefin-1, regulating intracellular cysteine protease activity, and possibly protection against extracellular proteolytic damage to the parasite''s intestinal and tegumental surface proteins. Considering inhibition kinetics and previously demonstrated immunomodulatory properties of cystatin in parasitic nematodes a comparable function of FgStefin-1 is suggested and is at present under investigation. [Copyright &y& Elsevier]
- Published
- 2009
- Full Text
- View/download PDF
43. Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens
- Author
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Jayaraj, Ramamoorthi, Piedrafita, David, Dynon, Kemperley, Grams, Rudi, Spithill, Terry W., and Smooker, Peter M.
- Subjects
- *
FASCIOLIASIS , *VACCINATION , *ANTIPARASITIC agents , *MICROBIAL virulence , *COMPLEMENTARY DNA , *VETERINARY parasitology , *PREVENTION - Abstract
Abstract: Liver flukes produce cathepsin B and cathepsin L in their excretory–secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin L''s are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination. [Copyright &y& Elsevier]
- Published
- 2009
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- View/download PDF
44. Fasciola gigantica and Schistosoma mansoni: Vaccine potential of recombinant glutathione S-transferase (rFgGST26) against infections in mice
- Author
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Preyavichyapugdee, Narin, Sahaphong, Somphong, Riengrojpitak, Suda, Grams, Rudi, Viyanant, Vitoon, and Sobhon, Prasert
- Subjects
- *
FASCIOLA , *SCHISTOSOMA mansoni , *TRANSFERASES , *LABORATORY mice - Abstract
Abstract: Recombinant Fasciola gigantica glutathione S-transferase (rFgGST26) was expressed in Escherichia coli. This protein had 86% and 56% sequence identity with 26kDa GST from Fasciola hepatica and Schistosoma mansoni, respectively. Polyclonal antibody raised in ICR mice against rFgGST26 recognized immunoblotted 26kDa native GSTs from F. gigantica and S. mansoni. rFgGST26 was used as a vaccine in combination with Freund’s adjuvant to evaluate the induction of immune responses and protection against F. gigantica and S. mansoni infection in mice. Mice were immunized via subcutaneous (s.c.), intramuscular (i.m.) or intradermal (i.d.) routes. Strong protection (77–84%) against F. gigantica was observed in all routes. Immunization via s.c. route induced immune response with IgG1 isotype predominating, while i.m. and i.d. routes resulted in mixed IgG1/IgG2a immune responses. Passive intraperitoneal transfer of IgG1 predominating antisera from s.c. rFgGST26-immunized donors to naïve recipient mice resulted in 47% protection against F. gigantica infection. This suggests that the mechanism of resistance depends on the presence of specific antibody against rFgGST26. Immunization with rFgGST26 via i.m. and i.d. routes resulted in significant cross protection (55%) against S. mansoni infection in the i.d. route with mixed IgG1/IgG2a response with IgG1 isotype predominating. This indicated that rFgGST26 is a good vaccine candidate against F. gigantica in mice and could also provide cross protection against S. mansoni. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
45. Comparative molecular analysis of two asparaginyl endopeptidases and encoding genes from Fasciola gigantica
- Author
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Adisakwattana, Poom, Viyanant, Vithoon, Chaicumpa, Wanpen, Vichasri-Grams, Suksiri, Hofmann, Annemarie, Korge, Günter, Sobhon, Prasert, and Grams, Rudi
- Subjects
- *
PEPTIDASE , *GENES , *FASCIOLA , *PARASITOLOGY - Abstract
Abstract: In this study we describe the first cysteine proteinases of the MEROPS Clan CD family C13 in Fasciola gigantica. Family C13 contains asparaginyl endopeptidases and glycosylphosphatidylinositol-anchor transamidases and is also called the legumain family due to the discovery of the first asparaginyl endopeptidase in a legume. The cDNAs encoding two asparaginyl endopeptidases, FgLGMN-1 and FgLGMN-2, were cloned and used for the analysis of nucleic acid and protein properties. The deduced amino acid sequences showed 47.4% identity to each other and from 42.2 to 51.1% identity to homologs of other trematode species. The catalytic site residues histidine, cysteine and preceding hydrophobic residues, characteristic for the cysteine proteinase families C11, C13, C14, and C25, were found conserved. Northern and reverse transcription PCR analyses demonstrated that the transcriptional products are present in metacercariae, juveniles and adults. RNA in situ hybridization and immunohistochemistry revealed that RNA and protein products of the two genes are specifically expressed in the intestinal epithelium of juveniles and adults. Immune sera of mice infected with F. gigantica reacted with immunoblotted, bacterially expressed recombinant proteins starting 4 weeks after infection. Polyclonal antisera raised against the recombinant proteins detected 40 and 30kDa antigens, respectively in crude worm protein extracts but not in the excretion–secretion products of adult parasites. Likewise, legumain-specific activity was found in crude worm protein extracts but not in excretion–secretion products. This study elucidates the molecular characteristics of these proteins in F. gigantica and demonstrates differences in the biology between Fasciola and Schistosoma which may prove useful for the development of vaccines against fasciolosis in domestic livestock. [Copyright &y& Elsevier]
- Published
- 2007
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46. An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes
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Vichasri-Grams, Suksiri, Subpipattana, Pornpimol, Sobhon, Prasert, Viyanant, Vithoon, and Grams, Rudi
- Subjects
- *
CALCIUM-binding proteins , *FASCIOLA , *IMMUNE serums , *SERUM - Abstract
Abstract: A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite''s excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite''s tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins. [Copyright &y& Elsevier]
- Published
- 2006
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47. Fischoederius elongatus (Poirier, 1883) Stiles & Goldberger, 1910, a cryptic species of pouched amphistome (Gastrothylacidae)?
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Watthanasiri, Pichanee, Geadkaew-Krenc, Amornrat, Smooker, Peter M., and Grams, Rudi
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- *
BIOLOGICAL classification , *CYTOCHROME oxidase , *MITOCHONDRIAL DNA , *SPECIES , *TRANSCRIPTOMES , *CATTLE parasites , *AMINO acid sequence - Abstract
SUMMARY: Transcriptome analysis of Fischoederius spp specimens from Thailand suggests a species complex resembling Fischoederius elongates. [Display omitted] • Analysis of Fischoederius spp COX1 sequences suggests a species complex. • Fischoederius spp transcriptome analysis shows significant differences. • Fischoederius elongatus might be a cryptic species. • Classical taxonomic samples are not directly linked to current molecular data. • The taxonomy of the genus Fischoederius should be revised. Rumen flukes in the genus Fischoederius are neglected foodborne parasites of cattle in Asia. Fischoederius elongatus , first described in 1883 from a sample collected in Indonesia is the type-species of the genus and is found from South to East Asia. In this study Fischoederius spp were collected from cattle in Thailand. The flukes resembled F. elongatus and images of 48 specimens were taken and their DNA was isolated. The mtDNA sequence of the cytochrome c oxidase subunit I (COX1) gene was amplified by PCR and used for restriction analysis with MseI. Nine restriction patterns (A–I) were observed and the COX1 mtDNA sequence for each pattern was determined. Phylogenetic analysis clustered the nine COX1 sequences into five groups with 4.6–9.6 % sequence differences between the groups. This is beyond intragenic variation observed for the COX1 gene in other organisms and suggested that the analyzed specimens represented several species. A comparative transcriptome analysis of specimens with COX1 MseI patterns A, C, E supported this finding. The observed median base differences, both absolute and relative, in the protein coding sequences of 999 orthologs were similar to those between distinct fruit fly species. It is proposed that the genus Fischoederius contains undescribed species that follow the classic description of F. elongatus. [ABSTRACT FROM AUTHOR]
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- 2021
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48. Microscopic and molecular epidemiology of gastrointestinal nematodes in dairy and beef cattle in Pak Chong district, Nakhon Ratchasima province, Thailand.
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Wangboon C, Martviset P, Jamklang M, Chumkiew S, Penkhrue W, Rangdist S, Jirojwong R, Phadungsil W, Chantree P, Grams R, Krenc D, Piyatadsananon P, and Geadkaew-Krenc A
- Abstract
Background and Aim: Gastrointestinal (GI) nematode infection remains an important problem in livestock, particularly cattle. The infection may lead to serious health complications and affect animal products. The objective of this study was to investigate GI nematode infection and its associated risk factors in dairy and beef cattle farmed in Pak Chong District of Nakhon Ratchasima Province, northeast Thailand., Materials and Methods: Fecal specimens were collected from 101 dairy cattle and 100 beef cattle. Formalin-ethyl acetate concentration techniques were used to process the samples and the samples were observed under a light microscope. Samples were subjected to molecular identification of specific genera using conventional polymerase chain reaction and DNA sequencing., Results: The overall prevalence of GI nematode infection was 33.3%. The strongyle nematode was the most significant GI nematode in this area with a prevalence of 28.4%. The prevalence of strongyle nematodes was 58.0% in beef cattle and only 7.9% in dairy cattle. Trichuris spp. was another nematode found in both types of cattle with an overall prevalence of 5.0% with 9.0% in beef cattle and 1.0% in dairy cattle. The results of the epidemiological study indicate that the age of cattle, food, water sources, farming system, and housing floor are the most important risk factors. Among the strongyle nematodes, Ostertagia spp. was the most prevalent (82.0%), followed by Haemonchus spp. (62.3%) and Trichostrongylus spp. (8.2%), respectively., Conclusion: Infection with GI nematodes still exists in this area, particularly in beef cattle. Our reported data may benefit local parasitic control policies in the future., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Wangboon, et al.)
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- 2024
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49. Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin.
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Phadungsil W and Grams R
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- Agglutination, Amino Acid Sequence, Animals, Bacteria cytology, Bacteria drug effects, Erythrocytes cytology, Erythrocytes drug effects, Fasciola chemistry, Fasciola genetics, Helminth Proteins chemistry, Helminth Proteins genetics, Helminth Proteins metabolism, Hemagglutination drug effects, Humans, Mannose-Binding Lectin chemistry, Mannose-Binding Lectin genetics, Mannose-Binding Lectin metabolism, Sequence Alignment, Streptococcus cytology, Streptococcus drug effects, Helminth Proteins pharmacology, Mannose-Binding Lectin pharmacology
- Abstract
The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.
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- 2021
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50. Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.
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Tran NTD, Ton Nu PA, Intuyod K, Dao LTK, Pinlaor P, Nawa Y, Choowongkomon K, Geadkaew-Krenc A, Kosa N, Grams R, and Pinlaor S
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- Amino Acid Sequence, Animals, Antibodies, Helminth, Cathepsin B chemistry, Cathepsin B immunology, Cysteine Proteases chemistry, Humans, Immunoglobulin G, Models, Molecular, Protein Conformation, Sensitivity and Specificity, Cysteine Proteases metabolism, Enzyme-Linked Immunosorbent Assay veterinary, Fasciola enzymology, Fascioliasis diagnosis
- Abstract
Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.
- Published
- 2019
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