Saengduen Moonsom’s research while affiliated with Mahidol University and other places

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Background One Health is defined as an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals and ecosystems; this approach attracts stakeholders from multiple sectors, academic disciplines, and professional practices. The diversity of expertise and interest groups is frequently and simultaneously framed as (1) a strength of the One Health approach in the process of understanding and solving complex problems associated with health challenges such as pathogen spillovers and pandemics and (2) a challenge regarding consensus on essential functions of One Health and the sets of knowledge, skills, and perspectives unique to a workforce adopting this approach. Progress in developing competency-based training in One Health has revealed coverage of various topics across fundamental, technical, functional, and integrative domains. Ensuring that employers value the unique characteristics of personnel trained in One Health will likely require demonstration of its usefulness, accreditation, and continuing professional development. These needs led to the conceptual framework of a One Health Workforce Academy (OHWA) for use as a platform to deliver competency-based training and assessment for an accreditable credential in One Health and opportunities for continuing professional development. Methods To gather information about the desirability of an OHWA, we conducted a survey of One Health stakeholders. The IRB-approved research protocol used an online tool to collect individual responses to the survey questions. Potential respondents were recruited from partners of One Health University Networks in Africa and Southeast Asia and international respondents outside of these networks. Survey questions collected demographic information, measured existing or projected demand and the relative importance of One Health competencies, and determined the potential benefits and barriers of earning a credential. Respondents were not compensated for participation. Results Respondents (N = 231) from 24 countries reported differences in their perspectives on the relative importance of competency domains of the One Health approach. More than 90% of the respondents would seek to acquire a competency-based certificate in One Health, and 60% of respondents expected that earning such a credential would be rewarded by employers. Among potential barriers, time and funding were the most cited. Conclusion This study showed strong support from potential stakeholders for a OHWA that hosts competency-based training with opportunities for certification and continuing professional development.

Purpose Blastocystis spp. is a cosmopolitan intestinal protozoa, infecting 30 - 60% population in the developing countries worldwide. Although often asymptomatic, chronic Blastocystis spp. infection is associated with irritable bowel syndrome. The parasite displays 17 genetically diverse subtypes (STs), exhibiting a wide host-range including human, ungulates, avian, and non-human primates. In areas where human and animal live closely together, this potentially results in zoonotic infection by Blastocystis spp. This study aims to identify Blastocystis spp. subtype distribution in human and domestic animals in an endemic area for intestinal parasitic infections at the Thai-Myanmar border. Methods & Materials Human and animal stool samples collected from six subdistricts of Tak province, Thailand, were microscopically screened for the presence of Blastocystis spp. The Blastocystis-positive samples were subjected to 18s small-subunit rRNA-based PCR amplification of the Blastocystis spp. and chain-termination sequencing. Neighbour-joining analysis was performed on the sequences for evolutional study and phylogenetic tree construction using previously published strains as reference. Results Forty-four samples were positive by microscopy for Blastocystis spp. and 27 samples were successfully cultured. Eight isolates from human and three from domestic animals (goats and buffalo) were PCR amplified. Multiple alignment of the DNA sequences with the subtype reference sequences and phylogenetic analysis of the 18s small-subunit rRNAs revealed ST3, a human specific subtype, as the most prevalent (6/11). Other subtypes were ST1 (1/11), ST2 (1/11), and ST13 (1/11). Two samples yielded dubious results, although one sequence from buffalo sample showed high similarity to ST15, previously reported in camels and gibbons. Two sequences of the human samples identified as ST1 (specific in human and ungulates) and ST2 (specific to human and avian) showed close resemblance to those of pigs and ducks, indicating possible zoonotic transmission risks. Conclusion We provided information on the Blastocystis spp. subtypes circulating in an endemic area at the Thai-Myanmar border. The high sequence identity of Blastocystis isolates between human and domestic animal samples warrants the possible zoonotic transmission of this parasite. Our results suggest the proper policy implementation regarding water-supply and animal waste-management to prevent the parasite transmission to human.

Background Tha Song Yang District located at the Thai-Myanmar border, contributes to the second highest cases of amoebic dysentery due to intestinal parasitic infections (IPI). However, there were limited disease prevalence data, specific surveillance system, and intervention available. Objective This study aimed to explore the epidemiological features of the IPIs and apply One Health (OH) approach to solve IPI-related problems. Methods Prevalence of asymptomatic infections in human and animals, yearly symptomatic cases, and associated risk factors were investigated. The OH intervention included improving the knowledge, attitude, and practice (KAP) of the community, microscopic diagnosis training, and stakeholder engagement for IPI prevention designs. Results The prevalence of asymptomatic cases was much higher than the symptomatic cases. Infective stages of the intestinal parasites were discovered in animal stool and water samples, indicating possible transmission routes. One year after the intervention, there were significant declines in asymptomatic IPIs and symptomatic cases of amoebic dysentery. Significant improvements in KAP and awareness regarding water and manure-waste management of the community were observed. Conclusion We reported the successful application of the OH intervention in reducing the IPI prevalence and mitigating disease-related risks. The intervention might be applied to address other infectious diseases in the future.

Leptospirosis is a zoonotic infectious disease caused by pathogenic Leptospira species. Virulence proteins have been shown to be key determinants of the pathogenesis of pathogenic Leptospira. A specific peptide at a mass-to-charge ratio of 7000 Da was identified in Leptospira whole cells using matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. This peptide was specifically present in pathogenic Leptospira and in clinical isolates. We report here the characterization of this specific peptide using a proteomics approach. This peptide was significantly matched to a hypothetical conserved L. interrogans protein (LA2458) with a calculated molecular weight of 7140.136 Da containing a tellurite-resistance domain at its C terminus (TerB-C). The amino acid sequences revealed the presence of hydrophobic transmembrane portions and two linear B-cell epitopes. Despite its low abundance, this synthetic peptide demonstrated dose-dependent cytotoxicity toward African green monkey kidney (Vero) cells via the apoptosis pathway. The concentration of the peptide 100 µM induced about 50% of cell death after a 24 h exposure. This peptide could be useful for the diagnosis of leptospirosis and the study of pathogenesis.

Extracellular matrix (ECM) plays key roles in shaping fates of stem cells, not only by providing a suitable niche but also by mediating physical and biochemical cues. Despite intensive investigations on regeneration, the roles of ECM in fate determination of stem cells in animals with great regenerative potency, such as planarian, have remained unclear. Here, we developed a method for decellularizing and isolating extracellular matrix from planarians. Although the isolated scaffold appears translucent, it contains all the internal features resembling those of the structure of intact planarians, and we thus called it the “ECM-body”. Nuclear staining demonstrated that the ECM-body contains very few or no remaining cells. Histological sections displayed well-preserved morphological integrity of the specimen. Scanning electron microscopy showed a porous surface on the ECM-body, potentially suitable for housing cells. Furthermore, our preliminary experiment suggested that ECM-body can be utilized as a biomimetic scaffold for cell culture as it may support survival of injected neoblasts.

Extracellular matrix (ECM) plays key roles in shaping fates of stem cells, not only by providing suitable niche but also by mediating physical and biochemical cues. Despite intensive investigations on regeneration, the roles of ECM on fate determination of stem cells in animal with great regenerative potency, such as planarian, remained unclear. Here, we developed a method to decellularizing and isolating extracellular matrix from planarians. Although the isolated scaffold appears translucent, it contains all the internal features, resembling the structure of intact planarian, and which we thus called ECM-body. Nuclear staining demonstrated that ECM-body contains very little or no cell remained. Histological sections displayed a well-preserved morphological integrity of the specimen. Scanning electron microscope showed porous surface on ECM-body, potentially suitable for housing cells. Furthermore, our preliminary experiment suggested that ECM-body can be utilized as biomimetic scaffold for cell culture as it may support survival of injected neoblasts.

Amoebiasis is a neglected protozoa infection affecting mostly developing countries and causing traveler’s diarrhea in developed countries. Despite its importance, epidemiological data were often inaccurate, leading to ineffective control programs. This problem stemmed from difficulties of accurate diagnosis, since the causative agent, Entamoeba histolytica, is indistinguishable by microscopy from closely related but less- or non-pathogenic Entamoeba species: E. moshkovskii and E. dispar. PCR-based molecular diagnosis is impractical for low-resource areas, due to costly equipment and professionals. Currently available antigen-detection immunodianostic platforms cannot differentiate E. histolytica from other Entamoeba species, and require fresh or unpreserved stool samples. This study aimed to overcome these limitations by assessing the performance of previously characterized Entamoeba species-specific monoclonal antibodies (mAbs) in detecting E. histolytica and E. moshkivskii trophozoites. Lowest detection limit of each mAbs was assessed against E. histolytica and E. moshkovskii cells by indirect ELISA. Specificity was tested against commonly found human intestinal protozoa. We also evaluated mAb reactivities against formalin-fixed Entamoeba cells. We found that our selected anti-E. histolytica and anti-E. moshkovskii mAbs detected as low as 100 and 10 of E. histolytica and E. moshkovskii cells, respectively. The mAbs exhibited no cross-reactivity towards closely related Entamoeba species and other intestinal protozoa. Interestingly, they also reacted with formaline-fixed cells. In the future, we will assemble them into an easy-to-use sandwich ELISA platform. We expect that this innovation might contribute to accurate differentiation of Entamoeba species, to reveal the true burden of these parasites and support successful control programs in the future.

Background Despite similarities in morphology, gene and protein profiles, Entamoeba histolytica and E. moshkovskii show profound differences in pathogenicity. Entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while E. moshkovskii causes only mild diarrhea. Extensive studies focus on roles of host immune responses to the pathogenic E. histolytica; however, evidence for E. moshkovskii remains scarce. Methods To study differences in host-antibody response profiles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with different sets of Entamoeba trophozoites as single species, mixed species and combinations. Results Mice prime-immunized with E. histolytica and E. moshkovskii combination, followed by individual species, exhibited higher IgG level than the single species immunization. Mice immunized with E. moshkovskii induced significantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interestingly, E. histolytica-specific anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no effect on cell adhesion. There was no significant effect of immunized sera on cytopathic activity and adhesion of E. moshkovskii toward both CHO and human epithelial human colonic (Caco-2) cell lines. Monoclonal-antibody (mAb) characterization demonstrated that 89% of E. histolytica-specific mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specific mAbs targeted plasma membrane proteins. Conclusions The present findings suggest that infection with mixed Entamoeba species or E. moshkovskii effectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difference of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifications of the amoebic parasites to escape from host immune system.

Background: Mosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni. Methods: In this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin. Results: Calcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa. Conclusions: Our results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.