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.

Evaluation of Immunodiagnostic Performances of Neospora caninum Peroxiredoxin 2 (NcPrx2), Microneme 4 (NcMIC4), and Surface Antigen 1 (NcSAG1) Recombinant Proteins for Bovine Neosporosis.

Bovine neosporosis is among the main causes of abortion in cattle worldwide, causing serious economic losses in the beef and dairy industries. A highly sensitive and specific diagnostic method for the assessment of the epidemiology of the disease, as well as it surveillance and management, is imperative, due to the absence of an effective treatment or vaccine against neosporosis. In the present study, the immunodiagnostic performance of Neospora caninum peroxiredoxin 2 (NcPrx2), microneme 4 (NcMIC4), and surface antigen 1 (NcSAG1) to detect IgG antibodies against N. caninum in cattle were evaluated and compared with that of the indirect fluorescent antibody test (IFAT). The results revealed that NcSAG1 had the highest sensitivity and specificity, with values of 88.4% and 80.7%, respectively, followed by NcPrx2, with a high sensitivity of 87.0% but a low specificity of 67.0%, whereas NcMIC4 showed sensitivity and specificity of 84.1% and 78.9%, respectively, when compared with IFAT. A high degree of agreement was observed for NcSAG1 (k = 0.713) recombinant protein, showing the highest diagnostic capability, followed by NcMIC4 (k = 0.64) and NcPrx2 (k = 0.558). The present study demonstrates that NcSAG1 is helpful as an antigen marker and also demonstrates the potential immunodiagnostic capabilities of NcPrx2 and NcMIC4, which could serve as alternative diagnostic markers for detecting N. caninum infection in cattle. These markers may find utility in future treatment management, surveillance, and risk assessment of neosporosis in livestock or other animal host species. Further research should be directed toward understanding the in vivo immune response differences resulting from immunization with both recombinant proteins.

Mass Spectrometry-Based Metabolomics Revealed Effects of Metronidazole on Giardia duodenalis.

Giardia duodenalis is a significant protozoan that affects humans and animals. An estimated 280 million G. duodenalis diarrheal cases are recorded annually. Pharmacological therapy is crucial for controlling giardiasis. Metronidazole is the first-line therapy for treating giardiasis. Several metronidazole targets have been proposed. However, the downstream signaling pathways of these targets with respect to their antigiardial action are unclear. In addition, several giardiasis cases have demonstrated treatment failures and drug resistance. Therefore, the development of novel drugs is an urgent need. In this study, we performed a mass spectrometry-based metabolomics study to understand the systemic effects of metronidazole in G. duodenalis. A thorough analysis of metronidazole processes helps identify potential molecular pathways essential for parasite survival. The results demonstrated 350 altered metabolites after exposure to metronidazole. Squamosinin A and N-(2-hydroxyethyl)hexacosanamide were the most up-regulated and down-regulated metabolites, respectively. Proteasome and glycerophospholipid metabolisms demonstrated significant differential pathways. Comparing glycerophospholipid metabolisms of G. duodenalis and humans, the parasite glycerophosphodiester phosphodiesterase was distinct from humans. This protein is considered a potential drug target for treating giardiasis. This study improved our understanding of the effects of metronidazole and identified new potential therapeutic targets for future drug development.

Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera.

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites.

Title: L'immunoprotéomique pour identifier chez Neospora caninum les antigènes spécifiques de l'espèce reconnus par les sérums de bovins infectés. Abstract: La néosporose bovine est une maladie préoccupante en raison de sa distribution mondiale et de son impact économique important par d'énormes pertes dans les industries laitières et de la viande. À ce jour, il n'existe aucun médicament chimiothérapeutique ou vaccin efficace pour prévenir la néosporose. Par conséquent, le contrôle de cette maladie dépend de tests de détection efficaces qui affecteraient les stratégies de gestion du traitement. Cette étude a été menée pour identifier les protéines immunoréactives spécifiques des tachyzoïtes de Neospora caninum reconnues par les sérums de bovins infectés par N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis et B. bigemina et par les sérums de bovins non infectés, à l'aide d'un gel d'électrophorèse bidimensionnel (2DE) combiné à l'immunoblot et à la spectrométrie de masse (LC-MS/MS). Parmi 70 spots protéiques ayant réagi avec tous les sérums infectés, 20 spots antigéniques spécifiques correspondant à 14 protéines antigéniques différentes ont été reconnus par les sérums positifs à N. caninum. Parmi ces antigènes immunoréactifs, les protéines impliquées dans la prolifération cellulaire et le processus d'invasion étaient hautement immunogènes, notamment la protéine de type HSP90, le micronème putatif 4 (MIC4), l'actine, le facteur d'élongation 1-alpha et la protéine à répétition de type armadillo/bêta-caténine. Fait intéressant, nous avons découvert un produit protéique sans nom, la protéine de rhoptries (ROP1), possédant une forte immunoréactivité contre N. caninum mais sans données disponibles sur sa fonction. De plus, nous avons identifié des antigènes à réaction croisée parmi ces parasites apicomplexes, en particulier N. caninum, T. gondii et C. parvum. Des protéines immunodominantes spécifiques de Neospora caninum ont été identifiées pour l'immunodiagnostic et le développement de vaccins. Les antigènes à réaction croisée pourraient être évalués comme candidats vaccins communs potentiels ou comme cibles médicamenteuses pour contrôler les maladies causées par ces parasites protozoaires apicomplexes.

Antibacterial Activities and Synergistic Interaction of Citrus Essential Oils and Limonene with Gentamicin against Clinically Isolated Methicillin-Resistant Staphylococcus aureus.

Citrus reticulata Blanco and Citrus aurantifolia are the edible plants which contain several biological properties including antibacterial activity. The aims of the present study were to determine the chemical compositions and evaluate antibacterial activities of citrus essential oils extracted from the fruit peels of C. reticulata (CREO) and C. aurantifolia (CAEO), alone and in combination with gentamicin, against a panel of clinically isolated methicillin-resistant S. aureus (MRSA) (n = 40) and methicillin-susceptible S. aureus (MSSA) (n = 45). Gas chromatography-mass spectrometry analysis revealed that 12 and 25 compounds were identified in CREO and CAEO with the most predominant compound of limonene (62.9-72.5%). The antibacterial activities were determined by agar disk diffusion and resazurin-based microdilution methods. The results found that almost all MRSA isolates were resistant to ciprofloxacin, erythromycin, and clindamycin, and some isolates were resistant to gentamicin. CREO and CAEO exhibited inhibitory effects toward clinical isolates (MIC: 1.0-32.0 and 8.0-32.0 mg/mL, respectively), with a similar trend to limonene (MIC: 1.0-32.0 mg/mL). However, the higher antibacterial effects were found in CREO and limonene when compared to CAEO (p < 0.01). In combination effect, the results showed the synergistic interaction of gentamicin with CREO and limonene on the MRSA and MSSA isolates (FIC indexes: 0.012-0.258 and 0.012-0.375), but that interaction of gentamicin with CAEO was observed only on MRSA (FIC index: 0.012-0.016). These findings demonstrated the potential of these citrus essential oils as natural antibacterial agents that may contribute to reduce the emerging of antimicrobial-resistant bacteria.

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.

Epidemiology and subtype distribution of Blastocystis in humans: A review.

Blastocystis is a commonly encountered gastrointestinal protozoan in humans and animals with uncertain pathogenicity. Despite its potential public health impact, epidemiological data regarding the prevalence and molecular subtype (ST) distribution of Blastocystis have been rarely reported. Among Blastocystis STs, ST1-ST4 are common in humans, including healthy and immunodeficient populations. According to the Chi-squared (χ2) association based on the data compiled for this cross-sectional study, the presence of ST1 is associated with asymptomatic infection, whereas the presence of ST4 is associated with symptomatic infection. However, cross-sectional studies cannot clarify the potential pathogenicity of Blastocystis, unlike in vivo and in vitro studies. Poor hygiene, poor sanitation and zoonotic transmission are possible factors associated with high Blastocystis prevalence, although this protozoan may be part of the normal healthy human gastrointestinal microbiota. This review covers the prevalence, STs and distribution of Blastocystis infection in humans. Thus, future epidemiological and subtyping studies could reveal new STs in humans as well as possible associations of STs with disease, drug resistance and related mechanisms such as protease activity. These associations with proper ST identification may facilitate the control of potential threats to host health, including the direct pathogenic effects of Blastocystis or alterations of the gastrointestinal microbiome.

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.

Prevalence and Subtype Distribution of Blastocystis Infection in Patients with Diabetes Mellitus in Thailand.

Diabetes mellitus (DM) is a major global public health problem with an increasing prevalence. DM increases the risk of infections caused by bacteria, fungi, viruses, and parasites. We examined the prevalence, subtypes, and risk factors of Blastocystis infection in patients with and without DM in central Thailand. Stool samples and questionnaires were obtained from 130 people in the DM group and 100 people in the non-DM group. Blastocystis infection was identified via a nested polymerase chain reaction and subtyped via sequencing of the partial small-subunit ribosomal RNA (SSU rRNA) gene. Analysis of potential risk factors was conducted via binary logistic regression. The overall prevalence of Blastocystis infection was 10.8%, including rates of 9% and 12.3% in the non-DM and DM groups, respectively. The most prevalent subtype was ST3, followed by ST1, and ST4. Factors that potentially increased the risk of Blastocystis infection include patients being >65 years old, the presence of DM, a DM duration of ≥10 years, a low level of education, and animal ownership. In conclusion, this is the first study of Blastocystis infection in DM, and a high prevalence was found among this population. Therefore, health education promoting sanitation and hygiene is necessary to reduce and prevent infection in the community.

Insecticidal and Histopathological Effects of Ageratum conyzoides Weed Extracts against Dengue Vector, Aedes aegypti.

Crude extracts and essential oils of A. conyzoides were tested with larva and adult stages of Ae. aegypti mosquitoes to determine their insecticidal properties. The crude extracts and essential oils came from three varieties of A. conyzoides (with white flowers, purple flowers, or white-purple flowers) and from two places on each plant (leaves and flowers), giving six types overall: leaf-white (LW); leaf-purple (LP); leaf white-purple (LW-P); flower-white (FW); flower-purple (FP); and flower white-purple (FW-P). Chemical constituents and components of the essential oils were identified using gas chromatography-mass spectrometry (GC-MS). Electron microscopic and histopathological studies were performed to determine the toxicological effects on mosquitoes in terms of morphological alterations. The six types of crude extracts exhibited no activity against individuals in the larval stages. However, six types of essential oils were effective against adult Ae. aegypti females. The mortality of adult Ae. aegypti females was higher from leaf extracts, particularly LP (median lethal dose, LD50 = 0.84%). The number of chemical constituents identified by GC-MS was high in flowers, especially W-P. Precocene I was the most abundant chemical component among the five types of essential oils, except in LP, in which precocene II was the most abundant. Histopathological alterations in adult Ae. aegypti females included compound eye degeneration, muscular damage with cellular infiltration, gut epithelial degeneration and necrosis, pyknotic nuclei in the malpighian epithelium and ovarian cell degeneration. FW and FP plant types exhibited the highest severity of histopathological alterations in mosquitoes compared with other plants, probably owing to the presence of monoterpene compounds in their tissues. The present study demonstrated LP plant extracts from A. conyzoides could be effective adulticides against adult Ae. aegypti. As natural products are biodegradable and exhibit low toxicity to mammalian and non-target organisms, they are suitable candidates for use in vector control programmes.

Efficacy of Ageratum conyzoides extracts against Giardia duodenalis trophozoites: an experimental study.

BACKGROUND: Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. METHODS: Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 µg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. RESULTS: LW-P and FP extracts showed high activity (IC50 ≤ 100 µg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 µg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW-P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 µg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. CONCLUSION: Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.

"Enterocytozoon bieneusi and Cryptosporidium: a cross-sectional study conducted throughout Thailand".

BACKGROUND: Enterocytozoon bieneusi and Cryptosporidium spp. are prevalent zoonotic parasites associated with a high burden among children. To date only limited molecular epidemiological data on E. bieneusi and Cryptosporidium spp. in humans living in Thailand has been published. METHODS: PCR-based tools were used to detect and characterize E. bieneusi and Cryptosporidium spp. The internal transcribed spacer (ITS) region of the rRNA gene was used to investigate E. bieneusi, and the small subunit (SSU) rRNA gene was used to investigate Cryptosporidium spp., and 697 fecal samples from villagers and school children in rural areas in Thailand were analyzed. RESULTS: The infection rates were 2.15% (15/697) for E. bieneusi and 0.14% (1/697) for Cryptosporidium spp. The prevalence of E. bieneusi was significantly high in Loei province. Sequence analysis indicated that the Cryptosporidium isolate was C. parvum. Nine E. bieneusi genotypes were identified, EbpC, Peru12, TMH6, TMH3, TMH7, H, D, and two novel genotypes TMLH1 and TMLH2. E. bieneusi prevalence was significantly higher in male participants than in female participants, and in children aged 3-15 years than in participants aged > 15 years. CONCLUSIONS: The prevalence, genotypes, and zoonotic potential of E. bieneusi were found to vary significantly high even in one country. Transmission routes and key animal carriers of E. bieneusi may be associated with differences in hygiene, sanitation, and cultural behaviors. Further molecular studies including longitudinal studies will be required to unveil epidemiological characteristics of these opportunistic intestinal protozoa in all over the countries.

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.

Blastocystis subtype 5: Predominant subtype on pig farms, Thailand.

Blastocystis is a unicellular protist most commonly detected in humans and a variety of animals. The predominant mode of its transmission is the fecal-oral route, but its zoonotic potential is not completely understood. The objective of this study was to determine the presence and genetic diversity of Blastocystis on pig farms in Nakhon Pathom Province, Central Thailand. A total of 154 human and 90 pig stool samples were collected and analyzed. Nested PCR detected Blastocystis in 35.55% of the pig samples and 6.49% of the human samples. Subtyping based on regions of the small-subunit ribosomal RNA (SSU rRNA) gene identified three Blastocystis subtypes in pigs and humans: ST1, ST3, and ST5. Blastocystis ST5 was the predominant subtype, followed by ST1 and then ST3. All the sequences from the Blastocystis-positive samples from both pigs and humans were closely related. This study reveals a possibility of low host specificity of Blastocystis STs (ST1, ST3 and ST5) on pig farms in Thailand. We tentatively suggest that close contact with or exposure to pig stools may be a significant source of Blastocystis detected in pig handlers. Further studies are required to confirm the zoonotic transmission of this organism in Thailand, because pigs may play an important role in the transmission of Blastocystis.

Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand.

Blastocystis is a common intestinal pathogen of humans and a variety of animals, with various host-specific subtypes. The aim of this study was to determine the prevalence and subtype distribution of Blastocystis in humans and domestic animals, Thailand. 113 stool samples were collected from pigs, goats, and cattle in Ayutthaya Province (AP; central Thailand) and 218 stool samples were collected from pigs, dogs, cats, chickens, and humans in Kanchanaburi Province (KP; western Thailand). Blastocystis was detected by nested PCR targeting the SSU rRNA gene. Subtypes were identified by DNA sequencing, and phylogenetic analysis was conducted. The overall prevalence of Blastocystis in animals was 76.1% (86/113) and 11.88% (12/101) in AP and KP, respectively, and the prevalence in humans was 12.82% (15/117) in KP. The prevalence of Blastocystis in the AP and KP pigs were 87.88% (29/33) and 20.37% (11/54), respectively. Blastocystis ST5 was the most abundant in pigs in both areas while Blastocystis ST10 and ST12 were most frequently found in cattle and goats. In addition, low percentage of Blastocystis ST1 and Blastocystis ST14 were found in pigs and goats, respectively. In this study, Blastocystis ST3, followed by ST2 and ST1 were predominantly found in humans. In conclusion, pigs may be a natural host of Blastocystis and this ST may be the pig-adapted ST in the studied areas, in this study.

Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis.

BACKGROUND: Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum. METHODS: LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand. RESULTS: Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697). CONCLUSIONS: This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.

Zoonotic potential of Enterocytozoon genotypes in humans and pigs in Thailand.

Enterocytozoon bieneusi is an opportunistic intestinal pathogen infecting humans and a variety of animals. Its mode of transmission and zoonotic potential are not completely understood. E. bieneusi has been frequently identified in pigs. The objective of our study was to investigate E. bieneusi in pigs and humans in Western and Central Thailand to determine its presence, genetic diversity, and zoonotic potential. A total of 277 human and 210 pig faecal samples were collected and analysed. E. bieneusi was found in 5.4% and 28.1% of human and pig samples, respectively, by nested PCR. Genotyping based on the internal transcribed spacer regions of the small subunit ribosomal RNA demonstrated three known genotypes (D, H, PigEb10) and eight novel genotypes (TMH1-8) in humans, and five known genotypes (D, EbpA, EbpC, H, O) and 11 novel genotypes (TMP1-11) in pigs. All known genotypes identified in humans and pigs had zoonotic potential. Further studies are needed to evaluate zoonotic risk of novel genotypes, as pigs may play an important role in the transmission of E. bieneusi.

Subtype Distribution of Blastocystis in Communities along the Chao Phraya River, Thailand.

Blastocystis is a common zoonotic enteric protozoan that has been classified into 17 distinct subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distributions of Blastocystis in villagers living along the Chao Phraya River, Ayutthaya Province, Thailand, and to assess the risk of zoonotic infection. In total, 220 stool samples were collected, and DNA was extracted. PCR and sequencing were performed with primers targeting the small-subunit ribosomal RNA (SSU rRNA) genes. Blastocystis was present in 5.9% (13/220) of samples, and ST3 (5.0%; 11/220) was the predominant subtype, followed by ST2 (0.45%; 1/220) and ST6 (0.45%; 1/220). Phylogenetic trees were constructed with the maximum-likelihood method based on the Hasegawa-Kishino-Yano + G + I model, neighbor-joining, and maximum parsimony methods. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. All the sequences of the Blastocystis-positive samples (KU051524-KU051536) were closely related to those from animals (pig, cattle, and chicken), indicating a zoonotic risk. Therefore, the villagers require proper health education, especially regarding the prevention of parasitic infection, to improve their personal hygiene and community health. Further studies are required to investigate the Blastocystis STs in the animals living in these villages.

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.