Development and utilization of a visual loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for rapid detection of Echinostomatidae metacercaria in edible snail samples.

Trematodes belonging to the family Echinostomatidae are food-borne parasites which cause echinostomiasis in animals and humans. This is a global public health issue, particularly in East and Southeast Asia. A method to detect the infective stage of Echinostomatidae species is required to prevent transmission to humans. In this study, a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was developed for visual detection of the metacercarial stage in edible snails of the genus Filopaludina from local markets in Thailand. The LAMP-LFD method can be performed within 70 min at a consistent temperature of 66 °C, and the results can be interpreted with the naked eye. The detection limits of the assay using Echinostoma mekongi, E. macrorchis, E. miyagawai and Hypoderaeum conoideum genomic DNA were equal between the four species at 50 pg/µL. A specificity evaluation demonstrated that the LAMP-LFD assay had no cross-reaction with another parasite (Thapariella species) or with the snail host species (Filopaludina martensi martensi, F. sumatrensis speciosa, and F. s. polygramma). Clinical test assessments were compared to microscopic examination in 110 edible snail samples. The clinical sensitivity and specificity of the tests were 84.62 % and 100 %, respectively, with a strong level of agreement based on the kappa statistic and the results of both methods were not significantly different (p > 0.05) per McNemar's test. The test successfully developed in this study may be useful for the detection of the metacercarial stage in edible snails for epidemiological investigations, control, surveillance, and to prevent future echinostomiasis health issues.

Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay.

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/µL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar's test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP.

Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive-negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.

Evaluation of semi-quantitative colorimetric assays based on loop-mediated isothermal amplification indicators by using image analysis.

Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-quantitative chromatic analysis has been used as an alternative method to differentiate between two colors and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and determined the optimal model between Red-Green-Blue (RGB) and Commission Internationale de l'Eclairage (CIE) Lab color spaces to establish a semi-quantitative colorimetric assay via image analysis by the ImageJ program for loop-mediated isothermal amplification (LAMP), using the dyes malachite green and phenol red. The semi-quantitative colorimetric assays using the color distance values of the CIELab color space (ΔEab) were more suitable than those using the RGB color space (ΔERGB) for chromatic differentiation between positive and negative reactions in both indicator dyes, demonstrating the feasibility of this assay to be applied in the detection of a wide range of pathogens and infectious diseases.

Feasibility of a DNA biosensor assay based on loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the visual detection of Ascaridia galli eggs in faecal samples.

Ascaridia galli is an important nematode that causes ascaridiasis in free-range and indoor system chicken farms. Infection with A. galli may damage the intestinal mucosa and inhibit nutrient absorption, leading to a reduced growth rate, weight loss and a decreased egg production. Consequently, A. galli infection is a significant health problem in chickens. In this study, we developed a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for the visual detection of A. galli eggs in faecal samples. The LAMP-LFD assay consists of six primers and one DNA probe that recognize the internal transcribed spacer 2 (ITS2) region; it can be performed within 70 min and the results can be interpreted with the naked eye. Using the LAMP-LFD assay developed in this study, A. galli DNA was specifically amplified without any cross-reactions with other related parasites (Heterakis gallinarum, Raillietina echinobothrida, R. tetragona, R. cesticillus, Cotugnia sp., Echinostoma miyagawai) and definitive hosts (Gallus gallus domesticus, Anas platyrhynchos domesticus). The minimum detectable DNA concentration was 5 pg/µl, and the detectable egg count was 50 eggs per reaction. The assay can be performed in a water bath, without the need for post-mortem morphological investigations and laboratory instruments. It is therefore a viable alternative for the detection of A. galli in chicken faeces and can replace classical methods in field screening for epidemiological investigations, veterinary health and poultry farming management.RESEARCH HIGHLIGHTSThis is the first study using the LAMP-LFD assay for Ascaridia galli detection.The results can be observed by the naked eye.The developed assay can be used to detect Ascaridia galli eggs in faecal samples.

Multi-detection for paramphistomes using novel manually designed PAR-LAMP primers and its application in a lateral flow dipstick (LAMP-LFD) system.

Loop-mediated isothermal amplification (LAMP) has been applied for the detection of various parasites, and its application in lateral flow dipstick (LFD) can improve the convenience of point-of-care diagnosis. A novel PAR-LAMP probe and primers were designed by manual selection from a region of low variation in the ITS-2 DNA sequence. Up to six species of rumen fluke were detected by LAMP and LAMP-LFD in this study. Target specificity and sensitivity were tested, revealing a high target specificity (accuracy) and a low limit of detection (sensitivity). Different target sensitivities of paramphistome were presented, including 5 pg for Gastrothylax crumenifer and Carmyerius sp.; 1 pg for Fischoederius elongatus, Orthocoelium parvipapillatum, and O. dicranocoelium; and 0.1 pg for Paramphistomum epiclitum. LAMP-LFD can detect a paramphistome egg even in contaminated in feces that was spiked with the egg under laboratory conditions. In addition, natural paramphistome infection in cattle from Surat Thani and Khon Kaen provinces, Thailand, was evaluated by detection of egg contamination in fecal specimens using PAR-LAMP primers. The PAR-LAMP detection result was also statistically evaluated by microscopic examination of feces. This study presents the application of novel manually designed primers in a LAMP-LFD system for improving performance in detection and diagnosis assays for paramphistomosis.

Novel high-performance detection of Raillietina echinobothrida, Raillietina tetragona, and Raillietina cesticillus using loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD).

Cestodes belonging to the genus Raillietina are a major veterinary health problem in the poultry industry, especially in chickens (Gallus gallus domesticus) and ducks (Anas playtrhynchos domesticus). In this study, loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was established and validated for the detection of R. echinobothrida, R. tetragona, and R. cesticillus in one reaction. The LAMP-LFD assay can be completed in 75 min under isothermal conditions at 66 °C and the results can be obtained by observation with the naked eye. This assay was very specific and had no cross-amplification with other closely related parasites (Cotugnia sp., Diorchis formosensis, Fimbriaria sp., Echinostoma sp., E. miyagawai, Hypoderaeum conoideum, Prosthogonimus cuneatus, and Ascaridia galli) or their definitive hosts (G. g. domesticus, A. p. domesticus). The sensitivity of the LAMP-LFD assay was detected with three Raillietina species at 0.5 ng, which was enough for gravid proglottid DNA detection. The accuracy test showed that the LAMP-LFD assay demonstrated accurate verification results when compared to morphological results. This is a novel LAMP-LFD assay that is highly specific and sensitive for the detection of Raillietina species. It can be applied to detection for epidemiological investigations, monitoring programs, surveillance, control, and to solve veterinary health problems for the poultry industry in Raillietina endemic areas.

Molecular classification of rumen fluke eggs in fecal specimens from Suphanburi Province, Thailand, based on cytochrome C oxidase subunit 1.

Rumen fluke infections have been known to cause paramphistomiasis in both wild and domestic animals worldwide. Occasionally, coinfections of rumen flukes (Carmyerius, Fischoederius, and Paramphistomum) with liver flukes (Fasciola) have been observed due to the similar life cycles that these two species share. This study involved an alternative approach that was developed to classify and distinguish rumen fluke eggs from other genera by using the polymerase chain reaction (PCR) method based on cytochrome c oxidase subunit 1 (COI). Thirty-eight fecal specimens of Bos taurus from Suphanburi Province, Central Thailand were examined using the formalin-ether sedimentation technique. PCR detection was then performed using COI-specific primers that were developed in this study. The results showed that this primer set can classify and distinguish the egg specimens into a separate clade of the genera comprising Gastrothylax, Carmyerius, Fischoederius, Paramphistomum, Explanatum, and Fasciola. Moreover, epidemiological mapping revealed coinfections of three genera of rumen flukes at some collection sites, leading to the need to further investigate Paramphistomoidea infection along with Fasciolidae infection within the endemic area. This data is important for monitoring the outbreak of these parasites in Suphanburi Province, Thailand. It can be applied for initiating surveillance programs of paramphistomiasis and fascioliasis in veterinary studies.

The study of Cytochrome B (CYTB): species-specific detection and phylogenetic relationship of Echinostoma revolutum, (Froelich, 1802).

Echinostoma revolutum is known as a significant intestinal trematode in various species of animals and humans. It presents complexities in terms of both the morphological and molecular biological data. This is the first study of the application of Cytochrome B gene (CYTB) as a target for studying the phylogeny and designing species-specific primer of E. revolutum. Adult trematodes were harvested from experimentally infected hamsters at 18 days of post-infection. Each worm was identified based on their morphological appearance. The novel CYTB primers were designed from other Echinostoma species to initially amplify CYTB region in E. revolutum. All sequence data of E. revolutum in five provinces of Central Thailand were used as the target for designing the species-specific primer for E. revolutum. The results revealed that CYTB gene can separate E. revolutum into two sister groups by geographical distribution, comprising the eastern and western area groups. Moreover, it also separates E. revolutum from other Echinostoma species, including two sibling species; E. caproni and E. paraensei. In addition, we developed the high performance species-specific primer of E. revolutum. It can detect DNA from a single egg, as well as cercaria, metacercaria and adult stages of this trematode with no cross-reactions to other trematodes and their hosts. Therefore, this research is a positive initial step for the future study of E. revolutum CYTB. The future studies based on this gene should be continued with all species in revolutum complex to overcome the problems of systemic classification that arise in this complex group.

The rapid detection method by polymerase chain reaction for minute intestinal trematodes: Haplorchis taichui in intermediate snail hosts based on 18s ribosomal DNA.

The minute intestinal trematode, Haplorchis taichui, is an important parasite species that can infect humans and other mammals. This study investigated the outbreak of H. taichui in thiarid snails in the lower part of the Chao Phraya Basin, Thailand by employing morphological and molecular-based methods. In development of a specific primer of H. taichui, the PCR reaction was conducted with no cross-reaction to their hosts and other related trematode species. The highest level of sensitivity that could be amplified was 0.50 ng/µl and this was detected with only one egg in the sample. In terms of the epidemic results, the parapleurolophocercous cercaria infected only two species of thiarid snails (Melanoides tuberculata and Tarebia granifera) with an overall prevalence of 3.80% (23/605). The process of molecular identification revealed positive results indicating that eleven from twenty-three of parapleurolophocercous cercariae specimens in the lower part of the Chao Phraya Basin were H. taichui. In conclusion, this study has developed a rapid detection method, which can discriminate H. taichui from other parapleurolophocercous cercaria in intermediate snail hosts with a high level of sensitivity. Moreover, the high proportion of H. taichui in parapleurolophocercous cercaria (47.83%) indicated that H. taichui was the dominant species of this cercarial type and could infect cyprinoid fish in the lower part of the Chao Phraya Basin leading to public health problems in this area. Thus, a specific primer could be useful in the detection and surveillance of H. taichui outbreaks in their hosts. Recognition of this has resulted in the creation of important prevention programs in these infected areas in the further study.

Morphological Characteristics and Phylogenetic Trends of Trematode Cercariae in Freshwater Snails from Nakhon Nayok Province, Thailand.

The prevalence of cercarial infection in freshwater snails and their evolutionary trends were studied in Nakhon Nayok province, Thailand. A total of 2,869 individual snails were examined for parasitic infections. The results showed that 12 snail species were found to host larval stages of trematodes with an overall prevalence of 4.7%. The infected specimens included 7 types at the cercarial stage; cercariae, megalurous cercariae, echinostome cercariae, furcocercous cercariae, parapleurolophocercous cercariae, virgulate cercariae, and xiphidiocercariae. Regarding molecular identification, ITS2 sequence data of each larval trematode were analyzed, and a dendrogram was constructed using the neighbor-joining method with 10,000 replicates. The dendrogram was separated into 6 clades (order/family), including Echinostomatida/Echinostomatidae, Echinostomatida/Philophthalmidae, Opisthorchiida/Heterophyidae, Plagiorchiida/Prosthogonimidae, Plagiorchiida/Lecithodendriidae, and Strigeatida/Cyathocotylidae. These findings were used to confirm morphological characteristics and evolutionary trends of each type of cercariae discovered in Nakhon Nayok province. Furthermore, this investigation confirmed that the ITS2 data of cercariae could be used to study on phylogenetic relationships or to determine classification of this species at order and/or family level when possible.

Epidemiological situation and molecular identification of cercarial stage in freshwater snails in Chao-Phraya Basin, Central Thailand

Objective: To investigate the prevalence of cercarial trematode infection in snails and to examine the reconstruction of the phylogenetic relationship to explain the molecular system of cercarial stage trematodes to estimate the infection rate of in the definite host from the Chao-Phraya Basin. Methods: The snails were collected from 10 provinces of the Chao-Phraya Basin, Thailand by stratified sampling method. The snails were examined for cercarial infection by the crushing method. All DNA specimens were amplified with internal transcribed spacer 3 (ITS3) and ITS4 primer based on PCR technique. The sequence data were aligned and used to reconstruct the phylogenetic tree by unweighted pair-group method with arithmetic means with 10 000 bootstraps. Results: The overall rate of cercarial infection was found to be 5.90%(122/2 067). Snails in the family Thiaridae were found to be in the highest prevalence followed by Lym-naeidae, Bithyniidae, Planorbidae, Viviparidae, and Ampullariidae, respectively, while the Buccinidae family (Clea helena) did not reveal any infections. The frequently found species of cercariae were parapleurolophocercous cercariae, cercariae and megarulous cercariae. The monophyletic tree separated the snails into five groups comprised of Heterophyidae, Strigeidae, Lecithodendriidae, Philophthalmidae and Echinostomatidae using the sequence of Angiostrongylus cantonensis as an out-group. Conclusions: This study was the first to report on cercarial infection in the Chao-Phraya Basin, Thailand. This revealed that a high variety of freshwater snails were infected by cercariae stage trematodes with a high prevalence. The sequence data of ITS2 can be used to investigate the phylogenetic relationships of trematodes at the family level and in each clade of different families separated by the definitive hosts.

Molecular isolation and characterization of a novel occlusion body protein gene from Penaeus monodon nucleopolyhedrovirus.

The full-length of the occlusion body (OB) protein gene of Penaeus monodon nucleopolyhedrovirus (PemoNPV) was successfully isolated. The OB gene sequence contained an open reading frame (ORF) of 1359 nucleotides encoding a protein of 452 amino acid residues with a predicted molecular mass of 50.6 kDa. A putative late promoter element, TAAG, was identified 72 nt upstream of the translation start site. The amino acid sequences of tryptic digested peptides of PemoNPV OB protein obtained from LC-MS analysis matched quite well with various regions of deduced amino acid sequences. Recombinant PemoNPV OB proteins specifically reacted with monoclonal antibodies to PemoNPV OB protein. After comparison with nucleotide database, the PemoNPV OB ORF demonstrated 67% identity to an uncharacterized ORF of a baculovirus pathogenic for Penaeus vannamei. However, comparison against protein databases revealed no significant homology to other known proteins. To our knowledge, this PemoNPV OB gene is the first isolated and characterized gene of nucleopolyhedrovirus from shrimp.

Preferential suppression of yellow head virus (YHV) envelope protein gp116 in shrimp that survive challenge with YHV.

The DNA sequence that encodes the first 406 amino acid residues at the N-terminus of yellow head virus (YHV) protein gp116, namely N/2 gp116deltaTM, and the DNA sequence that encodes the next 392 amino acid residues at the C-terminus of gp116 (without the transmembrane region), namely C/2 gp116deltaTM, were cloned into pGEX-6P-1 plasmid and expressed in E. coli. Both recombinant proteins were expressed, purified by SDS-PAGE and used to immunize mice. The mouse anti-recombinant N/2 gp116 and C/2 gp116 antisera bound specifically to both the recombinant proteins and to natural gp116 protein in YHV-infected haemolymph as shown by Western blotting and in tissues as shown by immunohistochemistry. Immunohistochemical localization of YHV using anti-gp116 antiserum or monoclonal antibodies specific to gp116 (V3-2B), gp64 (Y18) and p20 (Y19) revealed similar immunoreactivity patterns for all these reagents in muscle and mandibular tissue in shrimp showing gross signs of yellow head disease. However, in gill, hepatopancreas, lymphoid organ and thoracic ganglion tissues from experimental YHV-infected shrimp (Penaeus vannamei and Palaemon serrifer) that did not show signs of disease, immunoreactivity to gp116 was reduced or absent while that for gp64 and p20 remained intense. Thus, some shrimp species were able to selectively inhibit the synthesis of gp116 in a manner that was associated with absence of gross signs of disease.

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV).

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.

Polyclonal antibodies specific for VP1 and VP3 capsid proteins of Taura syndrome virus (TSV) produced via gene cloning and expression.

Capsid protein genes VP1 and VP3 of Taura syndrome virus (TSV) were cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant VP1 (rVP1) and recombinant VP3 (rVP3) were produced, purified by SDS-PAGE and used for immunization of Swiss mice for antisera production. Anti-rVP1 and anti-rVP3 antisera showed specific immunoreactivities to rVP1 and rVP3 proteins, respectively, by Western blot assay and also yielded good results for detection of TSV in various shrimp tissues by immunohistochemistry. This is the first step towards our target of preparing monoclonal antibodies specific to rVP1 and rVP3 for use in simple immuno-diagnostic test kits for TSV detection and identification.