Modeling impacts of climate change on the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand.

Global climate change is now regarded as imposing a significant threat of enhancing transmission of parasitic diseases. Maximum entropy species distribution modeling (MaxEnt) was used to explore how projected climate change could affect the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand. A range of climate variables was used: the Hadley Global Environment Model 2-Earth System (HadGEM2-ES) climate change model and also the IPCC scenarios A2a for 2050 and 2070. Occurrence data from surveys conducted in 2009 and 2014 were obtained from the Department of Disease Control, Ministry of Public Health, Thailand. The MaxEnt model performed better than random for O. viverrini with training AUC values greater than 0.8 under current and future climatic conditions. The current distribution of O. viverrini is significantly affected by precipitation and minimum temperature. According to current conditions, parts of Thailand climatically suitable for O. viverrini are mostly in the northeast and north, but the parasite is largely absent from southern Thailand. Under future climate change scenarios, the distribution of O. viverrini in 2050 should be significantly affected by precipitation, maximum temperature, and mean temperature of the wettest quarter, whereas in 2070, significant factors are likely to be precipitation during the coldest quarter, maximum, and minimum temperatures. Maps of predicted future distribution revealed a drastic decrease in presence of O. viverrini in the northeast region. The information gained from this study should be a useful reference for implementing long-term prevention and control strategies for O. viverrini in Thailand.

Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

Proteomic identification of peroxiredoxin 6 for host defence against Opisthorchis viverrini infection.

Opisthorchis viverrini infection causes opisthorchiasis and is a risk factor for cholangiocarcinoma via chronic inflammation. To investigate the mechanism of O. viverrini -induced liver disease, we applied a proteomic approach to examine alterations in hepatic protein levels in O. viverrini -infected hamsters. Two-dimensional gel electrophoresis (2DE) revealed that O. viverrini infection induced upregulation (1.5- to 4.3-fold) of 25 proteins and downregulation (1.5 to 2.5-fold) of 24 proteins compared with uninfected animals. Expression of proteins related to stress response, DNA replication and repair, and cell structure was significantly increased, whereas that of proteins associated with normal liver function, such as metabolism, blood volume maintenance and fatty acid cycle was decreased. Among the upregulated proteins, a 2.7-fold increase in peroxiredoxin 6 (Prdx6), an antioxidant protein, was confirmed by 2DE and immunoblot analysis, Western blot and quantitative PCR. Immunohistochemical analysis showed that Prdx6 expression was observed mainly in the cytoplasm of inflammatory cells. These results suggest that Prdx6 is important for host defence against O. viverrini infection. This study provides basic information for Prdx6 as a potential biomarker and therapeutic target for opisthorchiasis.

Identification of proteolytic bacteria from thai traditional fermented foods and their allergenic reducing potentials.

This study aimed to identify proteolytic bacteria from Thai traditional fermented foods and investigate their allergenic reducing potentials to wheat and milk allergens. Nine bacteria were isolated from fermented foods as follows: fermented soybean seeds (Thua Nao), fermented soybean paste (Thua Nao), wheat flour dough of steamed stuffed bun (Sa La Pao), and soaked rice from Thai fermented rice-noodle (Kha Nhom Jeen) processing. Both phenotypic and genotypic identifications were used in this study. It was found that all isolates were Gram-positive rods. Seven isolates were matched and identified as Bacillus subtilis by both techniques, and the remaining 2 isolates were phenotypically and genotypically identified as B. licheniformis and B. subtilis, respectively. The concentrated crude enzyme of B. subtilis DB and SR could reduce allergenicity of gliadin by hydrolyzing the allergenic gliadin fragments detected by immunoblotting. Furthermore, the enzyme of B. subtilis DB could also reduce allergenicity of beta-lactoglobulin (beta-LG) detected by hydrolyzing the major allergenic epitope of beta-LG at Gln(35)-Ser(36) position. B. subtilis DB and SR can be applied for the production of hypoallergenic wheat flour or milk food products.

Apoptosis-related gene expression in hamster opisthorchiasis post praziquantel treatment.

The aim of this study was to investigate the apoptosis-related gene expression in hamster opisthorchiasis after praziquantel treatment. Hamsters were infected with Opisthorchis viverrini metacercariae then treated with praziquantel. The expression of apoptosis-related genes [i.e., apoptosis gene Bcl-2-associated protein X (BAX), caspase 9, p53, and protein kinase B (PKB)] was detected by real-time reverse transcription polymerase chain reaction. Histopathological analyses of liver tissues were studies by staining the sections with hematoxylin and eosin using light microscopy. Apoptotic assay was used to localize the apoptotic cell death. The results show that BAX, Akt/PKB, p53, and caspase 9 expression level were significantly increased on day 30 post infection and at 6 h post treatment and gradually decreased nearly to the uninfected control and 24 h post treatment, perhaps due to a decrease in inflammatory cells. Apoptotic staining was positive reaction at inflammatory cells and nuclei of epithelial bile ducts. Although using praziquantel has an advantage in killing parasites, our results show the effect of praziquantel treatment from host immune response that induces increased apoptosis-related genes in the short term due to an increase in inflammatory cells surrounding the bile ducts.

Apoptosis-related gene expressions in hamsters re-infected with Opisthorchis viverrini and re-treated with praziquantel.

Our objective was to reveal whether host immune response in hamster opisthorchiasis post-praziquantel treatment could induce apoptotic cell death in inflammatory cells. We, therefore, investigated apoptosis-related gene expression in hamsters re-infected with Opisthorchis viverrini (OV) and re-treated with praziquantel. Hamsters were re-infected with OV metacercariae then re-treated with praziquantel. The expression of apoptosis-related genes (i.e. apoptosis gene Bcl-2 associated protein X [BAX], caspase 9, p53 and protein kinase B [PKB]) was detected by real-time reverse transcription-polymerase chain reaction. Histopathological analyses of liver tissues were performed by staining the sections with haematoxylin and eosin using light microscopy. The results show that BAX, Akt/PKB, p53 and caspase 9 expression levels were significantly increased on day 30 post-infection and at 6 h post-treatment and gradually decreased to a level near the uninfected control and at 24 h post-treatment, perhaps because of a decrease in inflammatory cells. Apoptotic cell death was observed at the nuclei of epithelial cells of the bile ducts and of T cells. Our results suggest that repeated infection with OV and re-treatment with praziquantel induces a host immune response that increases inflammatory cells, which in turn leads to increase, apoptosis-related gene expression in the short term post-treatment.

Thermally induced and developmentally regulated expression of a small heat shock protein in Trichinella spiralis.

A cDNA encoding a small heat shock protein of Trichinella spiralis, Ts-sHsp, was cloned and expressed and is herein characterized. This cDNA encoded a predicted protein of 165 amino acids, which had a high sequence identity in alpha crystallin domain with various small heat shock proteins of other organisms. A Western blot analysis indicated that anti-Ts-sHsp recombinant antibody recognized the protein of adults and larvae migrating at about 19 kDa. An in situ localization study showed the protein to be abundantly present in the body wall muscle cells, hypodermis, stichocytes, and esophagus of muscle larvae. The Ts-sHsp recombinant protein possessed chaperone activity to suppress the thermally-induced aggregation of citrate synthase. This sHsp was expressed at various developmental stages of T. spiralis, but at different levels. A high level was observed in mature muscle larvae (infective larvae), which was much higher than the levels seen in adults, newborn larvae, or immature muscle larvae. The expression of the sHsp gene was thermal inducible, thus responding to both cold (0 degrees C) and heat shock (43 degrees C) stress; however, at different patterns. The expression of Ts-sHsp increased gradually from 3 to 72 h after cold stress, while the expression was elevated to its highest after 3 h heat stress and then decreased. These results suggest that this small heat shock protein likely plays a role in the tolerance to both chemical and physical stresses, thereby enhancing the survival ability of Trichinella muscle larvae.

Involvement of the c-Ski oncoprotein in cell cycle arrest and transformation during nurse cell formation after Trichinella spiralis infection.

The role of c-Ski, an oncoprotein encoded by the oncogene, c-ski, in Trichinella spiralis-infected muscle tissues during nurse cell formation, was investigated by following the expression kinetics and distribution of c-Ski (both protein and mRNA) in the infected muscle cell, as well as the expression kinetics of the transforming growth factor beta (TGF-beta) signaling pathway factor genes (TGF-beta, Smad2 and Smad4) which cooperate with c-Ski. Immunohistochemical analysis using an anti-c-Ski antibody indicated that in the early stages of infection (13 and 18 days post-infection (p.i.)) the increased expression of the c-Ski protein was limited to the eosinophilic cytoplasm and not the enlarged nuclei or basophilic cytoplasm. At a later stage of infection (23 and 28 days p.i.) the c-Ski protein was limited to the enlarged nuclei in the basophilic cytoplasm, rather than the eosinophilic cytoplasm. At 48 days p.i., the c-Ski protein was barely detectable. Real-time PCR analysis showed that expression of the c-ski gene increased from 13 days p.i., reached a peak at 23-28 days p.i. and then decreased to a low level by 48 days p.i. Expression kinetics for the TGF-beta signaling pathway factor genes (TGF-beta, Smad2 and Smad4) were similar to that of c-ski. These findings provide evidence that the c-Ski protein is involved in nurse cell formation through the TGF-beta signaling pathway process in the host cell nucleus.

What is the role of p53 during the cyst formation of Trichinella spiralis ? A comparable study between knockout mice and wild type mice.

During the cyst formation of Trichinella spiralis, the infected muscle cell undergoes basophilic change and apoptosis, which results in nurse cell formation. This study revealed expression kinetics of some apoptosis genes such as p53 and its closely related genes (tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21, p21waf). RT-PCR (reverse transcription polymerase chain reaction) results showed that these genes were temporarily expressed in the infected muscles during the cyst formation period, but not in normal muscles (or very low if any), which suggested the involvement of these apoptosis genes in the nurse cell formation. Cysts and neighbouring muscle cells were separately collected and RT-PCR was performed, which suggested that p53 was expressed in the cysts. An immunocytochemical study showed that p53 was expressed in the nucleoplasm of basophilic cell in the cyst and Trichinella larvae, which suggested involvement of these apoptosis genes in the nurse cell formation. The same p53 expression kinetic study was performed on p53 knockout mice. The knockout mice did not express p53 genes, but expressed the other apoptosis genes in the same kinetics with only minor exceptions, suggesting that the expressions of these genes during the cyst formation were more or less p53-independent. There were no differences in the number and morphology of the cysts between the knockout mice and wild type mice. Thus apoptosis seen during the Trichinella cyst formation can be operated in the presence or absence of p53.

Tumor necrosis factor receptor-mediated apoptosis in Trichinella spiralis-infected muscle cells.

In order to reveal the mechanisms underlying nurse cell formation during Trichinella spiralis infection, the expression of the factors of tumor necrosis factor-alpha (TNF-alpha)/TNF receptor 1 (TNFR-1) signalling pathway mediating apoptosis was investigated. The analysed factors included TNF-alpha, TNFR-1, TNF receptor-associated death-domain (TRADD), caspase 3, caspase 8, TNF receptor associated factor-2 (TRAF2) and receptor interactive protein (RIP), all of which are involved in the TNF-alpha/TNFR-1 signalling pathway-mediated apoptosis. The quantitative RT-PCR indicated that the infected muscle tissues up-regulate the expression of pro-apoptosis genes (TNF-alpha, TNFR-1 and TRADD, caspase 3 and caspase 8), and anti-apoptosis genes (TRAF2 and RIP) at the beginning of cyst formation. The expression returned to the normal level after cyst formation. The quantitative RT-PCR analysis of mRNA from tissue samples isolated by laser capture micro-dissection confirmed that the up-regulation of these genes was restricted in infected muscle cells, was not in the inflammation cells around infected muscle cells nor in normal muscle cells. The in situ localization study of proapoptosis (TRADD, caspase 3) and anti-apoptosis gene products (TRAF2) indicated that these were expressed in the basophilic cytoplasm (infected muscle cell origin) of the nurse cells. Thus the present study suggests that the TNF-alpha/ TNFR-1 signalling pathway is involved in nurse cell formation.

In vitro antiparasitic activity of extracts of Cardiospermum halicacabum against third-stage larvae of Strongyloides stercoralis.

Extracts of Cardiospermum halicacabum, medicinal plant, were tested in vitro for their effectiveness against third-stage larvae of Strongyloidesstercoralis. Third-stage larvae of S. stercoralis were isolated from cultures of dog's feces using agar plate culture method. The larvae (1,000 larvae/ml), suspended in phosphate buffer saline solution, pH 7.4, were exposed to aqueous and alcohol extracts (2,000 microg/ml) of C. halicacabum at 37 degrees C with 5% CO2. Ivermectin (250 microg/ml) and piperazine (2,000 microg/ml) were also used as the reference drugs. The survival of Strongyloides larvae based on its motility was determined daily for 7 days. Strongyloides larvae were viable after contact with ivermectin, piperazine and C. halicacabum (aqueous and alcohol) solutions, but most of them were immobilized, after exposure to aqueous and alcohol extracts of C. halicacabum within 72 and 48 h, respectively, while ivermectin took from 72 to 144 h, and piperazine more than 7 days, to achieve the same rate of nonmotility. Clearly, the viability of S. stercoralis larvae was significantly reduced when exposed to extracts of C. halicacabum. Further study is needed on the antiparasitic activity of aqueous and alcohol extracts of C. halicacabum against S. stercoralis.

A spectrum of functional genes mobilized after Trichinella spiralis infection in skeletal muscle.

Trichinella spiralis infection causes the transformation of infected muscle cells, which leads to nurse cell formation. To search for the candidate genes responsible for nurse cell formation, cDNA microarray analysis of muscle tissues was performed before and after Trichinella infection. The Atlas mouse 1.2 cDNA expression microarray revealed the expression profiles of 1176 known genes. Out of these, 311 gene expressions were detected in normal and/or infected muscles. After the infection, 184 out of the 311 genes increased in expression by more than 3-fold. These included genes responsible for cell differentiation, proliferation, cell cycle and apoptosis. Thus this study suggested candidate genes for further investigation to dissect the molecular mechanisms of nurse cell formation.

Trichinella pseudospiralis infection is characterized by more continuous and diffuse myopathy than T. spiralis infection.

A time course study was performed to reveal the sequence of histopathology after Trichinella spiralis or T. pseudospiralis infection in mice. A cyst was formed in the former case by about 18 days post infection and prominent myopathy was restricted within the cyst. In the latter case, however, no typical cyst was formed, and myopathy spread diffusely over the infected muscle tissues occupying half the area of muscle sections. An electron microscope observation revealed that the disintegration of muscle cells was delayed in T. pseudospiralis infection than in T. spiralis infection. Quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that apoptosis-related genes were expressed for a longer term in muscles infected with T. pseudospiralis than in those with T. spiralis, although the same spectrum of genes are mobilized. Examined apoptosis-related genes included tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21 (WAF1), p21(waf) ; Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/ threonine protein kinase, PKB. Micro-dissection of the infected muscle tissue and subsequent RT-PCR confirmed that the expressions of these genes are restricted to tissue with myopathy. Thus, the expression of the apoptosis-related genes correlated with continuous and diffuse myopathy caused by T. pseudospiralis infection.

Expression of apoptosis-related factors in muscles infected with Trichinella spiralis.

We found that the expression of mitochondrial apoptosis related genes (Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/threonine protein kinase, PKB) is elevated in Trichinella spiralis-infected muscles during encapsulation. Micro-dissection of the capsule and subsequent reverse transcription polymerase chain reaction (RT-PCR) confirmed that the expressions of these genes are restricted to the nurse cell. Immunocytochemistry revealed that pro-apoptosis factor (BAX, Apaf-1 and Caspase 9) are predominantly expressed in the basophilic cytoplasm (infected muscle cell origin) and anti-apoptosis factor (PKB) in the eosinophilic cytoplasm (satellite cell origin) of the nurse cell. Electron microscopy revealed that the pre-existing mitochondria in the muscle cells became swollen and disappeared immediately after newborn larva invasion, but new mitochondria of smaller size appeared in the cytoplasm. Nuclear fragmentation and condensation were observed in basophilic cytoplasm which is known to die. Together, the results suggest that the infected muscle cells transform but die through the process of apoptosis which is triggered by factors from the newly formed mitochondria. The anti-apoptosis factor may help the eosinophilic cytoplasm with its survival to ensure nurse cell function.

Differences and similarities of nurse cells in cysts of Trichinella spiralis and T. pseudospiralis.

The nurse cell in the cyst of Trichinella spiralis comprises at least two kinds of cytoplasm, derived from muscle or satellite cells, as indicated by the pattern of staining using regular dye (haematoxylin and eosin, or toluidine blue), alkaline phosphatase (ALP) expression, acid phosphatase (ACP) expression and immunostaining with an anti-intermediate filament protein (desmin or keratin). Muscle cells undergo basophilic changes following a T. spiralis infection and transform to the nurse cells, accompanied by an increase in ACP activity and the disappearance of desmin. Satellite cells are activated, transformed and joined to the nurse cells but remain eosinophilic. The eosinophilic cytoplasm is accompanied by an increase in desmin and ALP expression but not an increase in ACP activity. Differences in the staining results for ALP or ACP suggest that the two kinds of cytoplasm have different functions. Trichinella pseudospiralis infection results in an increase of ACP activity at a later stage than T. spiralis. There is also a difference in the location pattern of ACP in the cyst of T. spiralis compared with T. pseudospiralis. In T. spiralis, ACP is diffused within the cell, but in T. pseudospiralis, ACP distribution is spotty corresponding to the location of the nucleus. Trichinella pseudospiralis infection is accompanied by a slight increase in ALP activity. Activated satellite cells following a T. pseudospiralis infection exhibit an increase in desmin expression. The present study therefore reveals that nurse cell cytoplasm differs between the two Trichinella species and between the two origins of cytoplasm in the cyst of T. spiralis.

Molecular expression and characterization of a homologue of host cytokine macrophage migration inhibitory factor from Trichinella spp.

A homologue of cytokine macrophage migration inhibitory factor (MIF) from complementary DNA (cDNA) of Trichinella spiralis and Trichinella pseudospiralis was expressed in Escherichia coli and characterized. The sequence analysis indicated that the predicted amino acid sequence has an identity of 57 and 44% with the MIF of nematodes Trichuris trichiura and Brugia malayi respectively, and 41 and 40% with that of a human and a mouse, respectively. The identity in sequences of cDNA and amino acids between T. spiralis and T. pseudospiralis was 91 and 86%, respectively. Western blot analysis showed that anti-MIF antibodies positively stained proteins from the extracts of adult worms or muscle larvae migrating at about 12.5 kDa (3 isoforms with isoelectric point 5.23, 5.72, and 6.29). Semiquantitative reverse transcriptase-polymerase chain reaction revealed that the gene was expressed in various developmental stages, including in adult worms, newborn larvae, precyst muscle larvae, and postcyst muscle larvae, although there was difference in the expression level among these stages. The immunohistochemical analysis showed the MIF exists in the muscle cells of the body wall and some stichocytes of larvae. Histopathology of T. spiralis-infected muscles revealed an accumulation of mononuclear cells around the worms, and immunocytochemical staining showed these cells were not macrophages. Mononuclear cells, including macrophages, were, however, observed in cardiac muscles where the parasite did not encyst. Macrophages accumulated around the Sephadex beads transplanted in mice subcutaneously, but this accumulation was profoundly inhibited when the beads were pretreated with MIF recombinant protein.

Molecular cloning and characterization of a novel protein of Trichinella pseudospiralis excretory-secretory products.

A novel excretory-secretory (ES) protein of Trichinella pseudospiralis was produced. A cDNA library was constructed from mRNA of muscle larvae at 30 days post infection (p.i.) and immunoscreened with the antibody against ES products. A clone, designated Tp22-3, contained a cDNA transcript of 815 bp in length with a single open reading frame which encoded 244-amino acids (28407 Da in the estimated molecular mass). A database search revealed that no sequences had a homology to this predicted protein. The recombinant protein was produced in an Escherichia coli expression system. Stage specific expression of this protein was suggested from the following experiments. An antibody against the recombinant protein could stain proteins migrating at about 28 kDa (which is the expected size from the sequence) on Western blotting of crude extracts or ES products from 30 days p.i. muscle larvae, but failed to stain any proteins in crude extracts from newborn larvae or 15 days p.i. muscle larvae. The antibody reacted to the stichocytes of larvae at 30 days p.i., but did not react to 15 days p.i. muscle larvae. The production of an mRNA transcript for Tp22-3 gene was restricted largely to the 30 days p.i. muscle larvae and adult worms.