Genetic diversity and host specificity of Blastocystis in reptiles, Eastern Thailand.

Blastocystis inhabits the digestive tracts of a diverse range of hosts. Transmission patterns, including host specificity, and the clinical and public health significance of Blastocystis in humans remain poorly understood. This study aimed to investigate the distribution and genetic diversity of Blastocystis in herbivorous and carnivorous reptiles in Eastern Thailand. A total of 501 faecal samples were collected from 363 iguanas, 79 bearded dragons, 50 tortoises, and nine snakes in an animal breeding farm in Chonburi Province, Eastern Thailand. Detection and differentiation of Blastocystis was based on amplification, sequencing, and phylogenetic analysis of specific small subunit (SSU) ribosomal RNA genes from faecal DNA extracted from the samples. Altogether 101/501 samples (20 %) were polymerase chain reaction (PCR) and sequencing-positive for Blastocystis, 90 (89 %) of which were from iguanas; the remaining positive samples were from African spurred tortoise (n=6), Bearded dragon (n=3), Leopard tortoise (n=1), and Red-footed tortoise (n=1). Phylogenetic analysis revealed that most of the Blastocystis sequences from iguanas were largely similar, and they were distinct from those of the tortoises. Subtype 17 was found in the three bearded dragons and likely reflected Blastocystis from prey animals. This is the largest survey of Blastocystis in reptiles to date. Remarkable differences in Blastocystis colonization rates and genetic diversity were observed between iguanas and other reptile orders, and what was considered Blastocystis colonization was only observed in herbivorous reptiles.

Microbiome analysis of thai traditional fermented soybeans reveals short-chain fatty acid-associated bacterial taxa.

Thua Nao is a Thai traditional fermented soybean food and low-cost protein supplement. This study aimed to evaluate the bacterial community in Thua Nao from northern Thailand and assess potentially active short-chain fatty acids (SCFAs)-related bacteria. Sixty-five Thua Nao consisting of 30 wet and 35 dried samples were collected from six provinces: Chiang Rai, Chiang Mai, Mae Hong Son, Lampang, Lamphun, and Phayao. Bacterial diversity was significantly higher in the wet samples than in the dried samples. The dominant phyla were Firmicutes (92.7%), Proteobacteria (6.7%), Actinobacteriota (0.42%), and Bacteroidota (0.26%). The genus Bacillus (67%) was the most represented in all samples. Lactobacillus, Enterococcus, and Globicatella were enriched in the wet samples. Assessment of the SCFA-microbiota relationships revealed that high butyrate and propionate concentrations were associated with an increased Clostridiales abundance, and high acetate concentrations were associated with an increased Weissella abundance. Wet products contained more SCFAs, including acetate (P = 2.8e-08), propionate (P = 0.0044), butyrate (P = 0.0021), and isovalerate (P = 0.017), than the dried products. These results provide insight into SCFA-microbiota associations in Thua Nao, which may enable the development of starter cultures for SCFA-enriched Thua Nao production.

Prevalence and Subtype Distribution of Blastocystis Isolated from School-Aged Children in the Thai-Myanmar Border, Ratchaburi Province, Thailand.

Blastocystis is one of the most common enteric protozoa that inhabits the intestinal tract of humans and different animals. Moreover, it has a worldwide geographic distribution. Its main mode of transmission is via the fecal-oral route. At present, 26 subtypes are widely distributed across both humans and animals. The current study aimed to determine the prevalence and subtype distribution of Blastocystis among school-aged children living on the Thai-Myanmar border, Ratchaburi province, Thailand. In total, 508 samples were collected from children at six schools. The prevalence of Blastocystis infection was amplified and sequenced in the 600 bp barcode region of the small-subunit ribosomal RNA (SSU rRNA). The overall prevalence of Blastocystis infection was 3.35% (17/508). ST3 (11/17) was the most predominant subtype, followed by ST1 (5/17) and ST2 (1/17). A phylogenetic tree was constructed based on the Tamura92+G+I model using the maximum-likelihood algorithm. Surprisingly, all sequences of the ST3-positive samples were closely correlated with the cattle-derived sequence. Meanwhile, all sequences of the Blastocystis ST1-positive samples were closely correlated with the human-derived sequence. Nevertheless, further studies should be conducted to validate the zoonotic transmission of Blastocystis. Based on our findings, personal hygiene and sanitation should be improved to promote better health in children in this area.

Molecular detection of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in school children at the Thai-Myanmar border.

Few data are available on the genetic identity of enteric protists Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in humans in Thailand. In this study, 254 stool samples were collected from primary school children from Ratchaburi Province at the Thai-Myanmar border and examined for Cryptosporidium spp., G. duodenalis, E. bieneusi and Cyclospora cayetanensis using PCR techniques. The genotype identity of the pathogens was determined by DNA sequence analysis of the PCR products. Cryptosporidium felis was found in 1 stool sample, G. duodenalis in 19 stool samples, and E. bieneusi in 4 stool samples. For G. duodenalis, sub-assemblage AII was the dominant genotype, but one infection with assemblage F was found. The E. bieneusi genotypes found included known genotypes D and J, and one novel genotype (HPTM1). Cyclospora cayetanensis was not detected in any samples. Results of the preliminary study indicate that children at the Thai-Myanmar border from Ratchaburi Province, Thailand are infected with diverse zoonotic genotypes of Cryptosporidium spp., G. duodenalis, and E. bieneusi.

Molecular identification of Pentatrichomonas hominis in animals in central and western Thailand.

BACKGROUND: Pentatrichomonas hominis inhabits the digestive tracts of several vertebrates, such as humans, monkeys, pigs, dogs, cats and rats. This protozoan was originally considered a commensal of the digestive tract but has subsequently been identified as a potential zoonotic parasite and a causative agent of diarrhoea. Molecular techniques are considered more sensitive and specific to detect P. hominis. This study aimed to determine the presence and genetic diversity of P. hominis in animals in Thailand. A total of 403 faecal samples were collected from 119 cats, 55 dogs, 73 goats, 35 monkeys, 55 cattle and 66 pigs, and the presence of P. hominis was determined using the nested polymerase chain reaction method. Sequence analysis of small-subunit ribosomal RNA genes was used to determine the genotype of the organism. RESULTS: Twenty-six samples (26/403, 6.45%) were positive for P. hominis. The highest prevalence was found in cats (21/119; 17.65%), followed by cattle (3/55; 5.45%) and dogs (2/55; 3.64%). Seven out of 26 nucleotides demonstrated 100% sequence identity with existing sequences; additionally, 16 novel sequence patterns were identified. All nucleotide sequences of P. hominis-positive samples were shown in the same branch with the previously described P. hominis sequences found in humans, dogs and goat. CONCLUSION: This is the first study on P. hominis infections in animals in Thailand. Our findings revealed that the prevalence of P. hominis was significantly higher in cats than in cattle and dogs. Cats were the main reservoir host; however, P. hominis can infect several kinds of animals. Therefore, the proper waste management of animals is necessary to reduce and prevent infection in the community.

Identification of Enterocytozoon bieneusi in goats and cattle in Thailand.

BACKGROUND: Enterocytozoon bieneusi has been increasingly reported to infect domestic animals and humans, with human infections primarily reported as zoonotic in origin. The aim of the present study was to determine the presence and genotype of E. bieneusi in humans and domestic animals in central Thailand by testing stool samples of 200 apparently healthy humans, 73 goats, 60 cattle and 65 pigs using nested-PCR/ sequence analysis based on the ITS region of SSU rRNA genes. RESULTS: E. bieneusi tested positive in 2 (1%) of the 200 stool samples collected from humans and 56 (28.3%) of the 198 stool samples collected from domestic animals. The highest prevalence of E. bieneusi was observed in pigs (39/65, 60%), followed by goats (14/73, 19.2%) and cattle (3/60, 5%). Seven novel E. bieneusi genotypes were identified, which were named GoatAYE1-4 and PigAYE1-3 and clustered in either zoonotic Group 1 or Group 2. Moreover, eleven previously described E. bieneusi genotypes were also identified (O, D, H, SX1, CHC8, CHG3, CS-10, SHZC1, LW1, WildBoar5, and EbpC). All novel genotypes exhibited zoonotic potential from a phylogenetic analysis of ITS region. CONCLUSION: Our data showed that the prevalence of E. bieneusi is low in apparently healthy individuals and higher in pigs than cattle and goats. This study provides baseline data useful for controlling and preventing E. bieneusi infection in farm communities, where pigs and goats appear to be the major reservoir of E. bieneusi. The results of our study support the view that E. bieneusi is a zoonotic pathogen that should be considered a potential public health threat.

Oyster is an effective transmission vehicle for Cryptosporidium infection in human.

OBJECTIVE: To determine the ability of oysters to trap and maintain viable Cryptosporidium oocysts, and the feasibility of Cryptosporidium multiplication in oysters' organs. METHODS: Seventy oysters were raised in experimentally seeded natural seawater for up to 3 months, with weekly oocysts inoculations. Cryptosporidium oocysts, viable and non-viable, as well as other stages were detected using two immunofluorescence vital staining techniques (Sporo-Glo and Merifluor(®)) with confocal microscopy. Viability rate at various times after inoculations were calculated. RESULTS: Cryptosporidium oocysts were found most concentrated in oysters' digestive organs than in gill and water inside the oysters. Oocysts numbers were 857.33 at 24 h after inoculation and strikingly decreased to 243.00 and 126.67 oocysts at 72 h and 7 days, respectively. The oocysts in oyster were also less viable over time; 70%, 60% and 30% viable at 24 h, 72 h and 7 days after inoculation, respectively. At 77 days, the number of oocysts was very low and none was found at 84 days onwards. Although some oocysts were ruptured with released sporozoites, there was no evidence throughout the study of sporozoites multiplication to indicate that oyster is a biological host. Despite the significant reduction in oocysts number after 7 days of inoculation, the remained viable oocysts can still cause cryptosporidiosis. CONCLUSION: The findings confirm that Cryptosporidium parvum does not multiply in oyster, and is therefore not a biological host. Nevertheless, the results suggest that oyster can be an effective transmission vehicle for Cryptosporidium oocysts, especially within 24-72 h of contamination, with viable oocysts present at up to 7 days post infection. Unless consuming well-cooked oyster dishes, eating raw oyster remains a public health concern and at least 3 days of depuration in clean sea water prior to consumption is recommended.

Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.

Cryptosporidium oocyst detection in water samples: floatation technique enhanced with immunofluorescence is as effective as immunomagnetic separation method.

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10(1), 10(2), and 10(3) per 10 microl were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10(2) per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

Transmission cycles of Giardia duodenalis in dogs and humans in Temple communities in Bangkok--a critical evaluation of its prevalence using three diagnostic tests in the field in the absence of a gold standard.

The prevalence and associated risk factors for Giardia duodenalis in canine and human populations in Temple communities of Bangkok, Thailand were determined by evaluating three common diagnostic methods utilised to detect Giardia, namely zinc sulphate flotation and microscopy, an immunofluoresence antibody test and nested polymerase chain reaction (PCR) based on the SSU-rDNA gene. The diagnostic sensitivity and specificity together with the negative and positive predictive values of each test were evaluated in the absence of a gold standard using a Bayesian approach. The median estimates of the prevalence of infection with G. duodenalis in dogs and humans in Thailand were 56.8% (95% PCI, 30.4%, 77.7%) and 20.3% (95% PCI, 7.3%, 46.3%) respectively. PCR and immunofluorescence antibody tests (IFAT) were the most accurate tests overall with diagnostic sensitivity and specificity of 97.4% (95% PCI, 88.5%, 99.9%) and 56.2% (95% PCI, 40.4%, 82.9%) for the PCR and 61.8% (95% PCI, 40.8%, 99.1%) and 94.7% (95% PCI, 87.4%, 99.1%) for IFAT respectively Three cycles, anthroponotic, zoonotic and dog-specific cycles of G. duodenalis were shown to be operating among the human and canine populations in these Temple communities in Bangkok, supporting the role of the dog as a potential reservoir for Giardia infections in humans.

PCR-based coprodiagnostic tools reveal dogs as reservoirs of zoonotic ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok.

A survey of gastrointestinal parasites of dogs and humans from temple communities in Bangkok revealed that 58% of dogs and 3.4% of humans, among those sampled, were infected with hookworms utilising faecal flotation techniques and microscopy. A previously established polymerase chain reaction (PCR)-RFLP approach was utilised to determine the species of hookworms infecting dogs found positive for hookworm eggs. Single infections with Ancylostoma ceylanicum and Ancylostoma caninum were recorded in 77% and 9% of hookworm positive dogs, respectively and mixed infections with both species of Ancylostoma were recorded in 14% of dogs. A single-step PCR for the multiplex detection of Ancylostoma species and Necator americanus DNA in human faeces was developed and applied to characterise the species of hookworms in microscopy positive individuals. Single infection with N. americanus was recorded in five and A.ceylanicum infection in two, out of seven individuals positive for hookworm. This study demonstrates that humans are at risk of acquiring infection with A. ceylanicum in communities where this species of hookworm is endemic in dogs.

Free-living ameba contamination in natural hot springs in Thailand.

Thermo tolerant free-living ameba, Naegleria spp and Acanthamoeba spp contamination in natural hot springs in Thailand were carried out from 13 provinces. The temperature of hot springs water varied from 28 degrees-65 degrees C and pH from 6-8. We found that 38.2 % (26/68) of water samples were positive, Acanthamoeba was 13.2% (9/68) whilst Naegleria was 35.3% (24/68). Contamination by free-living ameba in natural hot springs may pose a significant health risk to people who use such water for recreation activities.

Spa, springs and safety.

Natural mineral water has long been used worldwide for bathing and health purposes. At present, Thailand is famous for health spas and natural hot springs among local people and tourists. Due to possible risks of exposure to harmful agents, we studied hazardous pollutants at 57 natural hot springs from 11 provinces in northern, central, eastern and southern Thailand. Pathogenic, free-living amebae of the genera Naegleria and Acanthamoeba, which can cause central nervous system infection, were found in 26.3% (15/57) and 15.8% (9/ 57), respectively. Dissolved radon, a soil gas with carcinogenic properties, was present in nearly all hot springs sites, with concentration ranging from 0.87-76,527 Becquerels/m3. There were 5 water samples in which radon concentration exceeded the safety limit for drinking. Legionella pneumoniphila (serogroups 1, 3, 5, 6, 7 10 and 13) were found in samples from 71.9% (41/57) of studied sites. Because spas and natural springs are popular tourist attractions, health authorities should be aware of possible hazards and provide tactful measures and guidelines to ensure safety without causing undue alarm to foreign and Thai tourists.

Protozoal contamination of water used in Thai frozen food industry.

This study evaluated the prevalence of contamination of water that was used for food preparation. Since protozoal cysts can be found in small numbers in water, 1,000 liters of either untreated or treated water were filtered through activated carbon block filters (1 microm nominal porosity). Identification of protozoa was performed using specific monoclonal antibodies against Giardia and Cryptosporidium parasites followed by fluorescence microscopy. Twelve of 20 untreated water samples (60%) were found to be contaminated by Giardia cysts, with an average of 53.33 cysts/1,000 liters (geometric mean 39.43), whilst 7 samples (35%) were contaminated by Cryptosporidium oocysts, with an average of 28.57 oocysts/1,000 liters (geometric mean 26.92). Three samples of untreated water (15%) were positive for both organisms. In contrast, none of the treated water samples were contaminated.