Browsing by Author "Tadashi Itagaki"

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    Identification of Fasciola flukes in Thailand based on their spermatogenesis and nuclear ribosomal DNA, and their intraspecific relationships based on mitochondrial DNA
    (2012-12-01) Pannigan Chaichanasak; Madoka Ichikawa; Prasert Sobhon; Tadashi Itagaki; Iwate University; Mahidol University
    We analyzed 147 Fasciola flukes obtained from cattle in Thailand based on their spermatogenetic ability, and nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial nicotiamide adenine dinucleotide dehydrogenase subunit 1 (ND1) genes as molecular markers. One hundred twenty-eight flukes, which had abundant sperm in their seminal vesicles (spermic) and showed the PCR-RFLP pattern of F. gigantica in the ITS1, were accurately identified as F. gigantica. The other 19 flukes that had no sperm in their seminal vesicles were aspermic Fasciola sp. with the RFLP patterns identical to that of F. gigantica. Twenty-nine ND1 haplotypes (Fg-ND1-Thai 2-30) were distinguished in the 128 F. gigantica flukes and were divided into haplotypes unique to Thailand and those common to other countries, suggesting the possibility that ancestral haplotypes were introduced into Thailand. Three haplotypes (Fg-ND1-Thai 7, 9 and 27) appeared to be the major haplotypes found in F. gigantica from Thailand. Only one haplotype (Fg-ND1-Thai 1) was found in the 19 aspermic Fasciola sp. flukes obtained from geographical regions, and the nucleotide sequence of Fg-ND1-Thai 1 was identical to that of the aspermic Fasciola sp. from Japan, Korea, China, Vietnam and Myanmar, suggesting that they were descendants with a common provenance and expanded to these countries in the relatively recent past. © 2012 Elsevier Ireland Ltd.
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    Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice
    (2013-10-01) Narin Changklungmoa; Pornanan Kueakhai; Suda Riengrojpitak; Kulathida Chaithirayanon; Pannigan Chaichanasak; Narin Preyavichyapugdee; Pathanin Chantree; Veerawat Sansri; Tadashi Itagaki; Prasert Sobhon; Mahidol University; Mahanakorn University of Technology; Silpakorn University; Iwate University
    Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 μg of rFgLAP combined with Freund's adjuvant (n = 10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3 %, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat. © 2013 Springer-Verlag Berlin Heidelberg.
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    Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L
    (2015-01-01) Veerawat Sansri; Krai Meemon; Narin Changklungmoa; Pornanan Kueakhai; Pathanin Chantree; Pannigan Chaichanasak; Natcha Lorsuwannarat; Tadashi Itagaki; Prasert Sobhon; Mahidol University; Burapha University; Mahanakorn University of Technology; Iwate University
    © 2015 Elsevier Ltd. Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its efficacy is currently being studied in the larger economic animals.
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    Vaccine potential of recombinant cathepsin B against Fasciola gigantica
    (2013-09-01) Pathanin Chantree; Manussabhorn Phatsara; Krai Meemon; Pannigan Chaichanasak; Narin Changklungmoa; Pornanan Kueakhai; Natcha Lorsuwannarat; Kant Sangpairoj; Sineenart Songkoomkrong; Chaitip Wanichanon; Tadashi Itagaki; Prasert Sobhon; Mahidol University; Chiang Mai University; Iwate University
    In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals. © 2013 Elsevier Inc.
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    Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice
    (2015-10-01) Pornanan Kueakhai; Narin Changklungmoa; Pannigan Chaichanasak; Wipaphorn Jaikua; Tadashi Itagaki; Prasert Sobhon; Burapha University; Mahanakorn University of Technology; Mahidol University; Iwate University
    © 2015 Elsevier B.V. In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro- F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n= 10) by subcutaneous injection with 50. μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immuno-blotting to react with the native FgCatL1 in the extract of newly excysted juveniles (NEJ), 4-week-old juveniles and the ES products of 4 week-old juveniles. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune response, respectively, it was found that both Th1 and Th2 responses were significantly increased in rproFgCatL1- and rmatFgCatL1-immunized groups compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rmatFgCatL1-immunized group showed a significant decrease when compared to rproFgCatL1-immunized group, indicating that rmatFgCatL1-vaccinated mice had reduced liver parenchyma damage. The pathological lesions of liver in vaccinated groups were significantly decreased when compared with control groups. This study indicates that rFgCatL1 has a potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in ruminants.
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    Vaccine potential of recombinant saposin-like protein 2 against Fasciolosis gigantica in mice
    (2013-11-12) Pornanan Kueakhai; Narin Changklungmoa; Suda Riengrojpitak; Pannigan Chaichanasak; Krai Meemon; Kulathida Chaithirayanon; Pathanin Chantree; Veerawat Sansri; Tadashi Itagaki; Prasert Sobhon; Mahidol University; Burapha University; Mahanakorn University of Technology; Iwate University
    Saposin-like protein 2 (SAP-2) is a protein that adult of Fasciola spp. use to lyse plasma membrane of red blood cells, so that their contents can be digested by proteases for the parasites' nutrients. Thus SAP-2 is a plausible target for vaccination against these parasites. Recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was expressed in Escherichia coli BL21 (DE3). A vaccination was performed in ICR mice (n= 10) by subcutaneous injection with 50. μg of rFgSAP-2 combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 30 F. gigantica metacercariae by oral route. The percentages of protection of rFgSAP-2 vaccine against F. gigantica were estimated to be 76.4-78.5% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The antibodies in immune sera of vaccinated mice were shown by immuno-blotting to react with native FgSAP-2 in the extract of 2- and 4-week-old juvenile parasites. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, it was found that both Th1 and Th2 humoral immune response were significantly increased in rFgSAP-2 immunized group compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rFgSAP-2-immunized group showed no significant difference from those of the non-immunized and infected group, indicating that early juvenile parasites induced liver parenchyma damage, even though the numbers of worm recoveries were significantly different. This study indicates that rFgSAP-2 has a high potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in larger economic animals. © 2013 Elsevier Ltd.