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Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain.

The genotype of Toxoplasma gondii has been extensively studied to determine whether its characteristics may influence the course of toxoplasmosis. Recent genotyping studies in South America have found Toxoplasma hybrid isolates or atypical strains which were more virulent and highly diverse than North American and European species. Although, the high seroprevalence of Toxoplasma infection among pregnant women and HIV-infected patients from Songklanagarind Hospital, Songkhla, Southern Thailand has been previously reported, there is no reported study of human genotyping data on T. gondii strains. This is the first genetic typing of T. gondii isolates obtained from naturally infected cats in Thailand. Five chromosomal loci were analyzed by a nested PCR-RFLP technique to determine the genotype of 13 T. gondii isolates from cat feces gathered in the South of Thailand. The PCR-RFLP patterns of SAG1, SAG2-new, SAG3, BTUB and GRA6 markers resulted in five diverse genotypes: the type I (one isolate), type III (two isolates), type II or type III (one isolate), recombinant genotypes (two isolates) and atypical genotypes (two isolates). The presence of unusual genotypes may lead to new virulent traits associated with more severe forms of human Toxoplasma infections. More evaluations are needed before conclusions could be made as to whether the oocysts from cat feces play an important role in the severity of Toxoplasma infections.

Thai medicinal plants represent a rich source of potential anti-parasitic compounds. 1-hydroxy-2-hydroxymethylanthraquinone (CFQ) purified from Coptosapelta flavescens, a plant commonly used to expel intestinal worms, indicated potential anti-giardial agent as shown in a previous study. This study aims to investigate its mechanism of action. We assessed whether CFQ was involved as an inducer of apoptosis as well as having effects on the fine structure of Giardia intestinalis trophozoites. We observed the consequences of exposing G. intestinalis trophozoites to CFQ and metronidazole, both had an IC50 of 0.42μg/ml, after 6, 12 and 24h exposure. An apoptosis in trophozoite was confirmed by the AnnexinV-FITC assay and as viewed by flow cytometry. CFQ at its IC50 induced apoptosis as early as 6h after incubation while metronidazole produced little or no apoptosis at its IC50 value. Ultrastructural analyzes at 24h demonstrated that both CFQ and metronidazole induced several physical alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, ventral disc damage, electron dense precipitates in the nuclei, all of which were indicative of cell death. However, membrane rupture was found only in G. intestinalis exposed to CFQ and this proved the induction of apoptosis. Taken together, we have provided a mechanistic explanation of the action of CFQ against G. intestinalis trophozoites. These results have provided further evidence that CFQ is a new compound that has the potential for use to treat infections from G. intestinalis.

This study investigated the distribution of parasites as main contaminants in water environments of peninsular Malaysia (October 2011–December 2011) and the southeastern coast of Thailand (June 2012). Sixty-four water samples, 33 from Malaysia and 31 from Thailand, of various water types were examined according to U.S. Environmental Protection Agency guidelines. Drinking or household water types from both countries were free from parasitic contamination. The recreational/environmental (except a swimming pool in Malaysia) and effluent water types from these two countries were contaminated with waterborne parasites: Giardia (0.04–4 cysts/L), Cryptosporidium (0.06–2.33 oocysts/L), hookworm (6.67–350 ova/L), Ascaris (0.33–33.33 ova/L), and Schistosoma (9.25–13.33 ova/L). The most contaminated sites were recreational lake garden 3 in Malaysia and river 2 in Thailand. Higher concentrations of Giardia, Cryptosporidium, and hookworm were found in samples from Malaysia than in samples from Thailand. The presence of Giardia cysts showed a significant association with the presence of Cryptosporidium oocysts (P < 0.005).

One of the most important routes of transmission for Toxoplasma gondii infection is the ingestion of foods contaminated with cat feces containing sporulated oocysts. The diagnosis of T. gondii infection by fecal microscopy is complicated, as other similar coccidian oocysts are often present in the same fecal specimen. This study aimed to identify T. gondii oocysts in cat feces using a novel PCR technique. Feline fecal specimens (n = 254) were screened for coccidian oocysts by light microscopy using the Sheather's flotation method. PCR analysis performed on the same specimens targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) regions were used to confirm the presence of Toxoplasma oocysts. By light microscopy, 49/254 (19.3%) of specimens contained coccidian oocysts. PCR analysis demonstrated 2/254 (0.8%) and 17/254 (6.7%) positive results using Tox and ITS-1 primers, respectively. However, coccidian oocysts were not identified on microscopic examination of specimens that were PCR-positive by Tox primers. Coccidian oocysts were identified on microscopic examination of 6/17 (35.3%) of the PCR-positive fecal specimens using ITS-1 primers. The BLAST results of 16 ITS-1 sequences were identified as T. gondii (n = 12; 4.7%) and Hammondia hammondi (n = 4; 1.6%). There was slight agreement between the 529 bp and ITS-1 PCR results (κ = 0.148). This is the first report of the detection of Toxoplasma oocysts using PCR analysis on feline fecal specimens from Southern Thailand. The ITS-1 region has potential as an alternative marker to identify T. gondii oocysts in feline fecal specimens.

Cyclization of oxime with different aldehydes and ketones under basic condition led to the formation of new dioxazole derivatives and the structure was elucidated by spectral data. The effects of diaoxazoles on the inhibition of growth of Entamoeba histolytica and Giardia intestinalis in vitro have been determined, and selected compounds further investigated for their toxicity. SAR showed that the compounds with 5-nitrothiophene group at the 3-postion of the diaoxazole ring were more active than those with the p-toluene group at the same position. It is interesting to note that the compounds found active against E. histolytica were not found active against G. intestinalis. Toxicity studies showed that the compound 8 and 9 were non-toxic against Vero cell line ATCC CCL-81.

This study evaluated the anti-giardial activity of chloroform, methanol and water extracts of 12 medicinal plants (39 extracts), commonly used as self medication by AIDS patients in southern Thailand. The plant extracts and a standard drug, metronidazole, were incubated with 2x10(5) trophozoites of Giardia intestinalis per millilitre of growth medium in 96-well tissue culture plates under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and the minimum inhibitory concentration and the IC50 value for each extract was determined. The chloroform extracts from Alpinia galanga, Boesenbergia pandurata, Eclipta prostrata, Piper betle, Piper chaba, Zingiber zerumbet, and the methanol extracts from B. pandurata and E. prostrata were classified as "active", i.e. with an IC50 of100 microg/ml, whereas the chloroform extract from Murraya paniculata was classified as being "moderately active". This study shows that extracts from some medicinal plants have potential for use as therapeutic agents against G. intestinalis infections.

The anti-amoebic effects of crude methanol extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall against Entamoeba histolytica infecting the caecum of mice were studied. Caecal amoebiasis in mice was induced by injection of Entamoeba histolytica trophozoites directly into the caecum. The mice were then treated orally with the extract, a standard drug (metronidazole), or vehicle p.o. for five consecutive days, beginning 24 h after the infection and were examined on the sixth day. At a dose of 1000 mg/kg per day, the extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall had a curative rate of 100, 40 and 26%, respectively. At a concentration of 500 and 250 mg/kg/day, extract from Piper longum fruit was still effective in 93 and 46% of the cases, respectively, while extract from Piper sarmentosum root at a dose of less than 1000 mg/kg per day did not cure any mice from amoebiasis. Extract of Quercus infectoria nut gall at a concentration of 500 and of 250 mg/kg per day cured 26 and 13% of mice, respectively. Metronidazole at a concentration of 125 and of 62.5 mg/kg per day had a curative rate of 100 and 60%, respectively. The severity of caecal wall ulceration was reduced in mice which received the extract and metronidazole as compared to the control animals.

Background Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines. Methods A total of thirty three samples, each consisting of twelve liters, were collected and processed to obtain the sediment. Ten liters of sample each was processed to detect Cryptosporidium spp. and Giardia spp. using an immunomagnetic separation method prior to enumeration via fluorescence microscope. Meanwhile, the remaining two liters were cultured to detect Acanthamoeba and Naegleria through microscopy examination and polymerase chain reaction (PCR) analysis. Results Twelve samples (36.4%) from river (5), swimming pool (1), pond (3), rain tank (1), and natural lake (2) were positive for Cryptosporidium spp., 17 (45.5%) samples from river (9), pond (2), swimming pool (1), rain tank (1), and natural lake (4) were positive for Giardia spp. while, 13 (33.3%) samples from river (3), swimming pool (2), pond (2), dispenser (1), well (1), tap (2) and natural lake (2) were positive for Acanthamoeba spp. and 5 (18.2%) samples from river (1), natural lake (1), tap (1), dispenser (1) and mineral (1) were Naegleria spp. positive. Physical parameters such as temperature, conductivity, total dissolved solid (TDS), salinity, dissolved oxygen (DO), pH, and turbidity and chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite were also measured. The highest chemical contamination was observed at pond 2. A good correlation was observed between Giardia and nitrite (r = 0.736, p

Parasites cause a significant burden of disease globally and the majority of parasitic infections fall into the category of Neglected Tropical Diseases (NTDs). The emergence of parasitic diseases, including zoonotic infections, is associated with the process of rapid urbanization, exponential population growth, borderless trade between countries and climate change. To adopt new methods of control, a number of important issues must be addressed such as environmental-based interventions, novel drug discovery, the next generation sero-markers and development of new strategies for parasite control, and so forth. In this special issue on parasites: from source to vector and human, we have invited a few papers that address such issues. The first paper of this special issue addresses serine proteinase activities in excretory-secretory products of Toxocara canis second stage larvae (TES) related to the known sequences of TES components for example, TES26 and MUC4. The authors use protein modelling to identify molecules that can be incorporated into diagnostic and therapeutic techniques ultimately leading to better methods of control. The second paper on Chagas' disease describes extensively on aspects of transmission, association with pregnant women and congenital infection, clinical manifestations, diagnosis, treatment, control strategies, case follow-up and disease complications in Latin America. The third paper of this special issue presents a brief overview of the helminth-associated immune response, the currently available helminth secretome data, some major secretome-derived immunomodulatory molecules, their potential mode of actions and the applicability of helminth-derived therapeutic proteins in the treatment of allergic and autoimmune inflammatory disease. The fourth paper addresses the greater risk of exposure to tick borne pathogens (Anaplasma phagocytophilum and Ehrlichia chaffeensis) among rural residents using serological mass screening in China. Significant risk factors associated with infection are: having contact with animals, planting crops, having more employment time and having a history of fever. This study also proposes safety measures required to reduce zoonotic transmission. The fifth paper shows the effectiveness of different concentrations of two fractions of Curcuma longa cortex rich in turmerones and their respective liposomal formulations for treating promastigote forms of Leishmania amazonensis. The hexane fraction from the turmeric cortex, incorporated in liposomal formulation (LipoRHIC), presents a promising result for a new anti-leishmanial agent. The sixth paper uses climate modelling for global warming to indicate geographical spread, increasing number of seasonal generations and epidemiological changes of Dirofilaria spp. In addition to dirofilariosis, it also covers other mosquito-borne diseases in Russia and its neighbouring countries. The final paper of this special issue reports on the next generation sero-marker for toxoplasmosis with improved specificity and sensitivity using Toxoplasma gondii dense granular protein-5 (GRA5) on human serum samples. Veeranoot Nissapatorn Nongyao Sawangjaroen Rogan Lee Subhash Chandra Parija

The objective of this study was to assess the physico-chemical parameters and waterborne parasites in selected recreational lakes from Malaysia. Samples were collected from seven stations of Recreational Lake A (RL-A) and six stations of Recreational Lake B (RL-B). The samples were processed to detect the presence of Giardia spp. and Cryptosporidium spp. using immunomagnetic separation kit, helminth eggs or ova by bright field microscopy and Acanthamoeba spp. by cultivation in non-nutrient agar. Chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite and physical parameters such as dissolved oxygen, electrical conductivity, pH, salinity, temperature and total dissolved solid were also measured. Both lakes were freshwater with salinity ranging from 0.05 to 0.09 ppt. Most stations of these lakes were contaminated with Cryptosporidium spp., Giardia spp., Ascaris spp. and hookworm. Schistosoma spp. was found in RL-B only, while Acanthamoeba spp. was found in all stations. Of all sampling sites, station 5 of RL-B is the most contaminated. Linear regression and correlation analysis revealed that Giardia spp. and Schistosoma spp. showed a significant negative correlation with turbidity (p

The anti-amoebic activities of chloroform, methanol and water extracts from 12 Thai medicinal plants (39 extracts) commonly used by AIDS patients in southern Thailand were screened, at a concentration of 1,000 microg/ml, against Entamoeba histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro. The extracts were incubated with 2x10(5) E. histolytica trophozoites/ml of medium at 37 degrees C under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and scored (1-4) according to the appearance and numbers of the trophozoites. The extracts that caused inhibition were selected and retested using the same conditions but with concentrations that ranged from 31.25 to 1,000 microg/ml using E. histolytica strain HM1:IMSS, and the IC(50) values for each extract were calculated. The chloroform extracts from Alpinia galanga (IC(50) 55.2 microg/ml), Barleria lupulina (IC(50) 78.5 microg/ml), Boesenbergia pandurata (IC(50) 45.8 microg/ml), Piper betle (IC(50) 91.1 microg/ml) and Piper chaba (IC(50) 71.4 microg/ml) and the methanol extract from B. pandurata (IC(50) 57.6 microg/ml) were all classified as "active", i.e. with an IC(50) of less than 100 microg/ml, whereas those from Murraya paniculata (IC(50) 116.5 microg/ml) and Zingiber zerumbet (IC(50) 196.9 microg/ml) were classified as being "moderately active". The IC(50) of a standard drug, metronidazole, was 1.1 microg/ml.

The activities of n-hexane, dichloromethane and methanol extracts from five anti-diarrheic Thai medicinal plants, Acacia catechu (Fabaceae) resin, Amaranthus spinosus (Amaranthaceae) whole plant, Brucea javanica (Simaroubaceae) seed, Piper longum (Piperaceae) fruit and Quercus infectoria (Fagaceae) nut gall were tested against the in vitro growth of fresh isolates of the intestinal protozoan parasite, Blastocystis hominis. The extracts at concentrations that ranged from 62.5 to 2000 microg/mL, were incubated with several isolates of Blastocystis hominis for 48 h. The activities were classified as killed, inhibited, moderately inhibited and not-inhibited. Dichloromethane and methanol extracts from the Brucea javanica seed and a methanol extract from Quercus infectoria nut gall showed the highest activity. At a concentration of 2000 microg/mL, the three extracts killed 82, 75 and 67% of the Blastocystis hominis samples tested and inhibited 94, 100 and 76% of them, respectively. Metronidazole, used as a reference antiprotozoan drug, at a concentration of 40 microg/mL, killed 97% of the Blastocystis hominis isolates and inhibited all samples tested at concentrations that ranged from 1.25 to 20 microg/mL.

The hookworm Ancylostoma caninum induces human eosinophilic enteritis (EE), probably via allergic responses to its secretions. Cysteine and metallo-proteinases may be the components of these secretions that elicit hypersensitivity reactions. In order to characterize genes encoding cysteine proteinases (CP) secreted by A. caninum, an adult hookworm cDNA library was constructed and screened with a cloned fragment of a hookworm CP gene. This fragment was obtained using consensus oligonucleotide, CP-gene-specific primers in the polymerase chain reaction. cDNAs encoding two CPs were obtained from the library and sequenced. The first gene, AcCP-1, encoded a cathepsin B-like zymogen CP of 343 amino acids (aa), predicted to be processed in vivo into a mature CP of 255 aa. Closest nucleotide identities were to Haemonchus contortus cysteine protease (61%) and to human cathepsin B (60%). The second gene, AcCP-2, encoded a mature CP of 254 aa, that showed 86% identity to AcCP-1, and 58% and 47% identity to bovine cathepsin B and human cathepsin B, respectively. Rabbit antisera raised against recombinant AcCP-1 reacted with esophageal, amphidial and excretory glands in formalin-fixed, paraffin embedded sections of both male and female adult hookworms, and with an antigen of approx. 40 kDa in Western blot analysis of excretory/secretory products from adult hookworms. Together, these immuno-hybridization results strongly suggest that the CP encoded by the AcCP-1 gene is secreted by hookworms. These are the first reported CP genes from hookworms. Proteinases encoded by these genes may be responsible for the CP activity that we have shown previously to be secreted by adult A. caninum.

This paper reports the first survey in Australia to use faecal culture to detect Dientamoeba fragilis in patients with diarrhoea. Of 3 different protozoal culture media evaluated on a case of known infection, modified Boeck & Drbohlav's medium was the most suitable. The organism could be grown from faeces stored for up to 24 h at room temperature, but for only 10 h at 4 °C. Culture was then used, in combination with microscopy of smears fixed with polyvinyl alcohol and trichrome-stained, to examine single stool specimens from 260 consecutive patients with diarrhoea in the city of Brisbane. D. fragilis was detected in 4 (1·5%) specimens, only 2 of which were positive by microscopy. Other protozoa were found in 36 (13·8%) specimens: Blastocystis hominis in 28 (10·8%), Giardia duodenalis in 4 (1·5%), Endolimax nana in 3 (1·2%), and Entamoeba coli in one (0·4%). One strain of Dientamoeba was cryopreserved in liquid nitrogen and recultured successfully 10 d later. Culture was more sensitive than microscopy in diagnosing D. fragilis infection and the organism, of dubious pathogenicity, was not common in patients with diarrhoea in this community. Prevalence surveys of intestinal protozoan infections should use faecal culture, and specimens should be less than 12 h old and not refrigerated. Dientamoeba strains isolated by culture can be cryopreserved.

Human eosinophilic enteritis (EE) may result from hypersensitivity to the excretory/secretory (ES) antigens of adult Ancylostoma caninum. The origin of several antigens were identified by probing adult A. caninum with mouse monoclonal antibodies (MoAbs), sera from mice vaccinated with ES antigens and sera from human EE patients. Six MoAbs (AC/ES 1-6) were produced against ES antigens, two being IgG3 and four IgM. Western blots demonstrated four different antigen specificities: MoAb AC/ES 1 bound strongly to an ES product at about 30 kDa; AC/ES 2 recognized a broad band ranging from 50-200 kDa; AC/ES 3, AC/ES 5 and AC/ES 6 reacted at about 68 kDa, and AC/ES 4 at about 97 kDa. Sections of formalin-fixed, paraffin embedded adult A. caninum were then incubated with these MoAbs and immunostained by the peroxidase-anti-peroxidase (PAP) technique. The target epitope of MoAb AC/ES 1 was found mainly in the oesophageal, amphidial and excretory glands; AC/ES 2 reacted weakly with many structures in the sections; AC/ES 3, AC/ES 5 and AC/ES 6 were specific for excretory glands only, and AC/ES 4 bound to amphidial glands. Sera from immunized mice reacted with all three (especially the excretory) glands and the cuticle. In an indirect assay, worm sections probes with three human EE patient sera demonstrated maximal staining in the amphidial glands. Our findings confirm that ES products of A. caninum include immunogenic glandular secretions which may be involved in the pathogenesis of human EE.

Background Plasmodium knowlesi, a simian malaria parasite, has been reported in humans in many Southeast Asian countries. In Thailand, most of the limited numbers of cases reported so far were from areas near neighbouring countries, including Myanmar. Methods Blood samples collected from 171 Thai and 248 Myanmese patients attending a malaria clinic in Ranong province, Thailand, located near the Myanmar border were investigated for P. knowlesi using nested PCR assays. Positive samples were also investigated by PCR for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, and were confirmed by sequencing the gene encoding the circumsporozoite protein (csp). Results Two samples, one obtained from a Thai and the other a Myanmese, were positive for P. knowlesi only. Nucleotide sequences of the csp gene derived from these two patients were identical and phylogenetically indistinguishable from other P. knowlesi sequences derived from monkeys and humans. Both patients worked in Koh Song, located in the Kawthoung district of Myanmar, which borders Thailand. Conclusion This study indicates that transmission of P. knowlesi is occurring in the Ranong province of Thailand or the Kawthoung district of Myanmar. Further studies are required to assess the incidence of knowlesi malaria and whether macaques in these areas are the source of the infections.