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.

Tegumental surface change in Paramphistomum epiclitum caused by Bombax ceiba flowers and black pepper seed extract.

Rumen flukes, parasites of the superfamily Paramphistomoidea, are found in cattle rumen. Heavy infections can cause symptoms such as diarrhea, weight loss, and poor body condition, resulting in a decrease in milk and meat production. This study compares the tegumental surface change of Paramphistomum epiclitum as a response to ethanolic extracts of Bombax ceiba flowers and black pepper seeds. Adult flukes were subjected to various concentrations of crude extracts, including 12.5, 25, 50, 100, and 200 µg/mL for 12, 18, and 24 h incubation. Scanning electron microscopy (SEM) exhibited that the ethanolic extracts of both Bombax ceiba flowers and black pepper seeds caused tegumental surface changes in adult P. epiclitum. Based on the results, Bombax ceiba flower extract has anthelmintic activity, compared with black pepper seed extract, towards adult P. epiclitum due to the deformation of the tegument at lower concentrations than black pepper extract.

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.

A simple color absorption analysis of colorimetric loop-mediated isothermal amplification for detection of Raillietina spp. in clinical samples using a 3D-printed tube holder coupled with a smartphone camera and notebook screen.

A simple method has been developed for semi-quantitative analysis of the colorimetric output of loop-mediated isothermal amplification (LAMP) using a 3D-printed tube holder with a smartphone and notebook for the detection of Raillietina, which is the cause of Raillietiniasis affecting free-range chicken farming. In this method, a light is directed from a notebook screen to the LAMP products in the tube holder and the color absorption of the LAMP products is measured by using the appropriate smartphone application. It was found that the malachite green dye-coupled LAMP (MaG-LAMP) assay showed the highest sensitivity and specificity for detecting Raillietina without any cross-reaction with other related parasites and hosts. The limit of detection was 10 fg/µL of DNA. A total of 60 fecal samples were infectively confirmed by microscopic examination and the results of microscopy compared with those of MaG-LAMP and triplex PCR assays. Microscopy and MaG-LAMP based on the color absorption demonstrated high agreement in Raillietina detection with kappa = 1. Rapid, simple, cost-effective, and easy interpretation of colorimetric LAMP assays and their high sensitivity make them superior to PCR and morphological investigation, demonstrating the feasibility of this assay in point-of-care screening to support farm management and solve chicken health problems. Our study presents is an alternative diagnostic method using semi-quantitative analysis of colorimetric LAMP based on the differing solution color absorptions between positive and negative reactions for infectious disease diagnosis.

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.

Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM).

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1 pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.

Molecular detection of three intestinal cestode species (Raillietina echinobothrida, R. tetragona, R. cesticillus) from poultry in Thailand.

Cestodes belonging to the genus Raillietina are a major veterinary health problem affecting the poultry industry, particularly chickens (Gallus gallus domesticus) and ducks (Anas playtrhynchos domesticus). The traditional method for accurately detecting this cestode based on their morphological characteristics is rather difficult due to the large number of morphological similarities. Consequently, this study aimed to develop specific primers for R. echinobothrida, R. tetragona, and R. cesticillus detection that could be used to indicate epidemic areas for protection and infection control. Specific primers were manually designed based on the internal transcribed spacer 2 region and validated, establishing the optimal temperature, final concentration in PCR mixture, specificity, and sensitivity of each primer set. The results showed that the primers amplify specific species without cross-amplifying other parasites and hosts. The PCR products were about 473, 352, and 397 bp long for R. echinobothrida, R. tetragona, and R. cesticillus, respectively. The sensitivity test demonstrated that R. echinobothrida and R. cesticillus-specific primers detect a minimum of 5×10-2 ng DNA, while R. tetragona-specific primers detect a minimum of 0.5 ng genomic DNA. The specific primers successfully developed in this study might be useful for detecting cysticercoids in intermediate hosts or adult stages in poultry for epidemiological surveys, management and control of infection.RESEARCH HIGHLIGHTS This study established specific primers for Raillietina species detection.The ITS2 region is an effective molecular marker for Raillietina identification.

Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite perspective.

We investigated the prevalence, morphological characters and molecular classifications of trematode cercariae in freshwater snails randomly collected from 59 sampling localities in Bangkok from May 2018 to March 2019. We used a crushing technique to observe the cercarial stage inside each snail body and amplified the internal transcribed spacer 2 regions of cercarial DNA using polymerase chain reaction methodology. The associated phylogenetic tree was reconstructed using Bayesian inference analyses. A total of 517 of 15 621 examined snails were infected with trematode cercariae, and the infected snails were classified into 11 species of seven families with a 3.31% overall prevalence of the infection. The Bithynia siamensis siamensis snail displayed the highest prevalence of infection (16.16%), whereas the Physella acuta snail exhibited the lowest prevalence (0.08%) of infection. Eight morphological types of cercariae were observed. The highest prevalence of infection was observed in mutabile cercaria (1.86%). Based on molecular investigations, the phylogram revealed eight cercaria types assigned to at least nine digenean trematode families, of which five belong to groups of human intestinal flukes. Although, with the exception of schistosome cercaria, trematode cercariae are not known to directly damage humans, understanding the general biology of trematode cercariae (including diversity, distribution, infection rates and host range) is important and necessary for the prevention and control of parasitic transmission that impacts aquatic cultivations, livestock farming and human health.

Infections of Digenetic Trematode Metacercariae in Wrestling Halfbeak, Dermogenys pusilla from Bangkok Metropolitan Region in Thailand.

This study aimed to investigate metacercarial infections in the wrestling halfbeak, Dermogenys pusilla, collected from Bangkok metropolitan region of Thailand. A total of 4,501 fish from 78 study sites were commonly examined with muscle compression and digestion methods (only head part of fish) during September 2017 to July 2018. The overall prevalence of metacercarial infection was 86.1% (3,876/4,501 individuals), and the mean intensity was 48.9 metacercariae per fish infected. Four species, i.e., Posthodiplostomum sp., Stellantchasmus falcatus, Cyathocotylidae fam. sp., and Centrocestus formosanus, of digenetic trematode metacercariae (DTM) were detected. The prevalences were 65.8%, 52.0%, 2.1%, and 1.2%, respectively and their mean intensities were 23.1, 51.6, 1.4, and 3.2 per fish infected, respectively. The seasonal prevalences were 81.0% in winter, 87.8% in summer and 87.4% in rainy, and the mean intensities were 38.9, 46.6, and 55.2 metacercariae per fish infected, respectively. Conclusively, it was confirmed that the wrestling halfbeak play the role of second intermediate hosts of 4 species of digenetic trematodes including S. falcatus and Posthodiplostomum sp. in Bangkok metropolitan region. And then the metacercariae of C. formosanus and Cyathocotylidae fam. sp. are to be first found in the wrestling halfbeak by this study.

Is species identification of Echinostoma revolutum using mitochondrial DNA barcoding feasible with high-resolution melting analysis?

The taxonomic evaluation of Echinostoma species is controversial. Echinostoma species are recognized as complex, leading to problems associated with accurate identification of these species. The aim of this study was to test the feasibility of using DNA barcoding of cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) conjugated with high-resolution melting (HRM) analysis to identify Echinostoma revolutum. HRM using COI and ND1 was unable to differentiate between species in the "revolutum complex" but did distinguish between two isolates of 37-collar-spined echinostome species, including E. revolutum (Asian lineage) and Echinostoma sp. A from different genera, e.g., Hypoderaeum conoideum, Haplorchoides mehrai, Fasciola gigantica, and Thapariella anastomusa, based on the Tm values derived from HRM analysis. Through phylogenetic analysis, a new clade of the cryptic species known as Echinostoma sp. A was identified. In addition, we found that the E. revolutum clade of ND1 phylogeny obtained from the Thailand strain was from a different lineage than the Eurasian lineage. These findings reveal the complexity of the clade, which is composed of 37-collar-spined echinostome species found in Southeast Asia. Taken together, the systematic aspects of the complex revolutum group are in need of extensive investigation by integrating morphological, biological, and molecular features in order to clarify them, particularly in Southeast Asia.

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.

Prevalence of Centrocestus formosanus Metacercariae in Ornamental Fish from Chiang Mai, Thailand, with Molecular Approach Using ITS2.

The prevalence of Centrocestus formosanus metacercariae was investigated in ornamental fish purchased from a pet shop in Chiang Mai, Thailand, including Carassius auratus (goldfish), Cyprinus carpio (Koi), Poecilia latipinna (Sailfin Molly), Danio rerio (Zebrafish), and Puntigrus tetrazona (Tiger barb). The parasite species was identified by the morphology of worms as well as by a molecular approach using ITS2. The results showed that 50 (33.3%) of 150 fish examined were infected with the metacercariae. The highest prevalence was found in C. auratus (83.3%), and the highest intensity was noted in C. carpio (70.8 metacercariae/fish). The most important morphological character was the presence of 32-34 circumoral spines on the oral sucker. The phylogenetic studies using the rRNA ITS2 region revealed that all the specimens of C. formosanus in this study were grouped together with C. formosanus in GenBank database. This is the first report on ornamental fish, C. carpio, P. latipinna, D. rerio, and P. tetrazona, taking the role of second intermediate hosts of C. formosanus in Thailand. Prevention and control of metacercarial infection in ornamental fish is urgently needed.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand.

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

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.

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.

The determination and relationship of four coexisting paramphistomes in perspective of integrative taxonomic investigation.

Co-infections with Orthocoelium species and other paramphistomes were found in different ruminant hosts from two provinces of Thailand. Whilst O. parvipapillatum coexisted with Paramphistomum epiclitum in the same cattle (Bos taurus) from Pathum Thani Province, Thailand, O. dicranocoelium and Fischoederius elongatus were found in water buffalo (Bubalus bubalis) from Chiang Mai Province. Morphological, histological, and tegumental surface features of both Orthocoelium species were intensively investigated for species differentiation. Statistical analysis of eight morphometric ratios presented morphological differences for three paramphistomes in the Paramphistomidae family and some relationships among paramphistomes in different definitive hosts. The genetic relationships of the co-infecting paramphistomes were investigated using p-distance and phylogenetic tree analyses. Genetic variations in the Orthocoelium co-infecting paramphistomes, P. epiclitum and F. elongatus, were calculated and compared to DNA sequence alignments based on internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) DNA markers. In addition, the phylogenetic tree constructions from both DNA markers and their concatenated sequence (ITS2 + COI) were used for species confirmation and the presentation of genetic relationships between co-infecting paramphistomes and other paramphistomes. This study improves the basic taxonomical description and understanding of parasite-parasite and host-parasite interactions from the perspectives of morpho-histological, morphometric, and genetic variation in co-infecting paramphistomes and Orthocoelium species in different hosts.

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.

High-performance triplex PCR detection of three tapeworm species belonging to the genus Raillietina in infected poultry.

Chickens and ducks are important sources of essential proteins and nutrition for global consumption, especially their eggs and meat. Tapeworm infections in chickens and ducks are the cause of serious poultry health and economic problems in the processing of livestock and food production systems. Raillietina are cosmopolitan in distribution and are possibly the most common tapeworm parasites. There are three important species regarding avian infection, with different pathogenicity, including Raillietina echinobothrida, R. tetragona, and R. cesticillus. Co-infection diagnosis of these tapeworms using morphological analysis can be performed, but this is time-consuming and complicated. Therefore, this study aimed to develop a triplex PCR for the detection and discrimination of three Raillietina species. The triplex PCR assay specifically amplified target DNAs with no inter-specific interference and produced a specific band for each species. According to the specificity test, there was no cross-amplification with the DNA template of related parasites and their hosts. The lowest detectable DNA concentrations were evaluated and provided sensitivities of 0.5 pg/µL for R. echinobothrida, 5 pg/µL for R. tetragona, 50 fg/µL for R. cesticillus, and 5 pg/µL for the combination of DNA from all three species. Simultaneous detection limits of egg capsules and gravid proglottids was also performed, with and without feces. The interference of feces in the reaction was related to a decrease in sensitivity, but simultaneous detection of three Raillietina species in amounts lower than one gravid proglottid and ten egg capsules was still successful. Thus, this study is the first triplex PCR assay for Raillietina detection and can be utilized as an alternative diagnostic tool for the detection and discrimination of R. echinobothrida, R. tetragona, and R. cesticillus infection in poultry through the verification of fecal specimens. In addition, it could improve the performance of specific treatments and promote veterinary healthcare.

Complex morphological characterization and morphometric-molecular discrimination of two paramphistome species co-infecting cattle, Orthocoelium sp. and Paramphistomum epiclitum.

Co-infection by two paramphistome species, Orthocoelium sp. and Paramphistomum epiclitum, is found in cattle in Thailand. The morphological features of these and other paramphistomes under a light microscope are similar, resulting in misidentification and misdiagnosis. We classified these paramphistomes into three morphological variation types, namely Orthocoelium sp., P. epiclitum MV1 (immature), and P. epiclitum MV2 (matured). Ten morphological characteristics were investigated, and the values were transformed into 25 ratio criteria for statistical investigation. Morphometric analysis can classify the variation of these specimens using differences in the bifurcal level, the vitellaria starting level, the starting level of the anterior testis, and the center level of the posterior testis positions by body length ratios. These ratios can separate the samples into three morphologically different groups, whereas molecular analysis based on the nuclear internal transcribed spacer 2 (ITS2) region and the mitochondrial cytochrome c oxidase subunit I (COI) gene could only distinguish two specific groups. In addition, the Orthocoelium specimen, related to O. dicranocoelium and O. parvipapillatum according to morphological and histological analysis, was monophyletic grouped via ITS2 analysis. Our study provides a scientific basis for the taxonomic classification and clustering of morphologically varying species, improving the identification, detection, and diagnosis of co-infecting paramphistomes.