Coproprevalence, seroprevalence, and geographic distribution of Fasciola spp. infection in beef and dairy cattle in Pak Chong highland, Nakhon-Ratchasima Province, Northeast Thailand.

Fasciola spp. is a major livestock parasite, especially in cattle, water buffalo, sheep, and goats. Infection reduces animal productivity, e.g., meat, dairy, wool and might cause death. In Thailand, reports of Fasciola spp. infection in livestock, especially dairy and beef cattle, are sparse. Pakchong district in Nakhon Ratchasima province is one of Thailand's largest farming areas for dairy and beef cattle, but the prevalence of Fasciola spp. infection has never been reported in this district. The landscape of this area is mainly a hilltop plateau with many water sources suitable for the development of lymnaeid snails, the intermediate host of Fasciola spp., which are essential for the parasite life cycle. This study surveyed the copro- and seroprevalence of Fasciola spp. infection in dairy and beef cattle farmed in Pakchong district by microscope-based examination, PCR, and indirect ELISA. Associated risk factors and geographic information data were collected and analyzed. Paired stool and serum samples were collected from 102 dairy cattle and 99 beef cattle from April to November 2021. Sample analyses demonstrated a high prevalence of Fasciola spp. infection, especially in beef cattle. The overall copro-prevalence was 5.97%, with 0.99% in dairy cattle and 11.11% in beef cattle. The overall seroprevalence was 23.88%, with 2.94% in dairy cattle and 45.45% in beef cattle. Moreover, the data indicated that infection status was not correlated with animal sex and age whereas consumption of natural grasses, water resources, housing floor, and farming system were significant risk factors. Data analysis by a geographic information system (GIS) demonstrated that an associated risk could be farmed in lowering areas, especially in Chan Thuck, Nong Sa Rai, and Khlong Muang subdistricts. In conclusion, this study reports the prevalence of Fasciola spp. infection in cattle in a major farming area of Thailand which could be beneficial for designing parasite control policies in this region as well as adapting this knowledge to other Fasciola spp. endemic areas.

Cystatins from the Human Liver Fluke Opisthorchis viverrini: Molecular Characterization and Functional Analysis.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

Current prevalence and geographic distribution of helminth infections in the parasitic endemic areas of rural Northeastern Thailand.

BACKGROUND: Helminth infection is a global health issue that not only causes acute helminthiasis but long-term infection may lead to complicated symptoms as well as severe complications. The World Health Organization cooperated with the Ministry of Public Health in many countries, particularly where high prevalence, spending a lot of resources for limiting the infection. In Thailand, the incidence of parasitic helminth infections was continuously declined in the last few decades according to several campaigns for parasitic elimination. However, the rural community in the northeast of Thailand where the highest prevalence of the country still needs to be monitored. This present study aims to report the current prevalence of parasitic helminth infections in Nakhon Ratchasima and Chaiyaphum provinces where sharing a huge area of the northeastern region of Thailand but only a few studies have been published. METHODS: The stool specimens were collected from 11,196 volunteers and processed by modified Kato-Katz thick smear, PBS-ethyl acetate concentration techniques, and PCR. The epidemiological data were collected, analyzed, and used for generating of parasitic hotspots. RESULTS: The results indicated that O. viverrini remains the major parasite in this area with a total prevalence of 5.05% followed by Taenia spp., Hookworms, T. trichiura, and Echinostoma spp., respectively. Mueang district of Chaiyaphum province has the highest prevalence especially O. viverrini with a prevalence of 7.15% that higher than the latest national surveillance. Interestingly, the prevalence of O. viverrini was hugely reported (more than 10%) in five subdistricts. The geographic localization of O. viverrini infections revealed that a lot of water reservoirs such as the lakes or branches of the river in the two-most prevalent subdistricts. Our finding indicated that gender and age were insignificantly different. CONCLUSION: This finding suggested that the parasitic helminth infection in the rural areas of northeast of Thailand remains high and the housing location is a major contributing factor for the parasitic infection.

Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L).

(1) Background: Opisthorchis viverrini is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still required. Immunodiagnosis seems promising, but attempts to generate monoclonal antibodies have not yet been successful. This study aims to develop a single-chain variable antibody fragment (scFv) against Rhophilin-associated tail protein 1-like (ROPN1L), the sperm-specific antigen of adult O. viverrini, which has not been reported elsewhere. (2) Methods: The target epitope for phage screening was L3-Q13 of OvROPN1L, which showed the highest antigenicity to human opisthorchiasis analyzed in a previous study. This peptide was commercially synthesized and used for phage library screening. The isolated phage was produced in a bacterial expression system and tested for specificity in vitro and in silico. (3) Results: One of fourteen phages, named scFv anti-OvROPN1L-CL19, significantly bound to rOvROPN1L compared with non-infected hamster fecal extracts. This phage clone was successfully produced and purified using Ni-NTA chromatography. Indirect ELISA demonstrated that scFv anti-OvROPN1L-CL19 has a high reactivity with O. viverrini-infected hamster fecal extracts (12 wpi, n = 6) in comparison with non-infected hamster fecal extracts (0 wpi, n = 6), while the polyclonal rOvROPN1L antibodies did not show such a difference. Molecular modeling and docking confirmed our in vitro findings. (4) Conclusion: scFv anti-OvROPN1L-CL19 could be used as an effective material for developing O. viverrini-immunodiagnostic procedures in the future.

Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs.

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection.

Improvement of a PCR-based method for the detection of Opisthorchis viverrini eggs in human stool samples by targeting internal transcribed spacer-2 (ITS-2), cytochrome oxidase subunit 1 (cox1), and cytochrome b (cyb).

Opisthorchis viverrini infection causes various complications in patients, ranging from asymptomatic to severe chronic disease including cholangiocarcinoma (CCA). O. viverrini is endemic in Southeast Asia and acting as a risk for CCA. Early diagnosis of O. viverrini infection can reduce the number of CCA cases. The routine diagnosis for opisthorchiasis is direct wet smear, sometimes coupled with concentration techniques, which has limitations when investigating light infection or if done by laboratorians with lack of experiences. PCR-based methods have been established for the detection of O. viverrini egg DNA from stool samples, but have never fully succeeded for light infections when compared to wet smear concentration techniques. This study aims to improve the PCR-based method for detection of O. viverrini eggs in stool samples by targeting the genes ITS-2, cox1, and cyb. The results reveal higher sensitivity than conventional concentration techniques, with all newly designed primers. ITS-2 has an overall sensitivity of 76.9% with 66.7% in the samples with < 50 EPG, while cox1 has shown 96.2% overall sensitivity and 94.1% in the same EPG intervals. Interestingly, the new pointing target, cyb, has shown 100% sensitivity in all egg intervals in this study, particularly for light infections (EPG less than 100). No cross-reactivity was found in Taenia spp., Trichuris trichiura, Ascaris lumbricoides, Capillaria philippinensis, and hookworm. The procedure is convenient, with shorter steps compared to previous reports, and it appears appropriate for use in the diagnosis of light infection with O. viverrini. These three genes are good candidates for use in PCR-based detection of the parasite eggs. Further testing with a larger cluster of samples is however necessary.

Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin.

The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.

Evaluation of Rhophilin Associated Tail Protein (ROPN1L) in the Human Liver Fluke Opisthorchis viverrini for Diagnostic Approach.

Tegumental and excretory-secretory proteins are reported as diagnostic antigens for human opisthorchiasis. Rhophilin associated tail protein1-like (OvROPN1L) protein of Opisthorchis viverrini sperm tail showed potential as a diagnostic antigen. The OvROPN1L recombinant fragments were assayed for diagnostic antigenicity for human opisthorchiasis using indirect ELISA. The strongest antigenic region was a N-terminus peptide of M1 - P56. One synthetic peptide (P1, L3-Q13) of this region showed the highest antigenicity to opisthorchiasis. Sera from other parasitic infections including Strongyloides stercoralis, hookworm, Taenia spp, minute intestinal flukes, Paragonimus spp showed lower reactivity to P1. Peptide P1 is located in the disordered N-terminus of ROPN1L supporting its suitability as linear epitope. In the Platyhelminthes the N-terminal sequence of ROPN1L is diverging with taxonomic distance further suggesting that peptide P1 has potential as diagnostic tool in the genus Opisthorchis/Clonorchis. It should be further evaluated in combination with peptides derived from other O. viverrini antigens to increase its diagnostic power.

Similarity of a 16.5kDa tegumental protein of the human liver fluke Opisthorchis viverrini to nematode cytoplasmic motility protein.

Opisthorchis viverrini is the causative agent of human opisthorchiasis in Thailand and long lasting infection with the parasite has been correlated with the development of cholangiocarcinoma. In this work we have molecularly characterized the first member of a protein family carrying two DM9 repeats in this parasite (OvDM9-1). InterPro and other protein family databases describe the DM9 repeat as a protein domain of unknown function that has been first noted in Drosophila melanogaster. Two paralogous proteins have been partially characterized in the genus Fasciola, Fasciola hepatica TP16.5, a novel tegumental antigen in human fascioliasis and, recently F. gigantica DM9-1, a parenchymal protein with structural similarity to nematode cytoplasmic motility protein (MFP2). In this study, we show further evidence that this family of trematode proteins is related to MFP2 in sequence and structure. Soluble recombinant OvDM9-1 was used for structural analyses and for production of specific antisera. The native protein was detected in soluble and insoluble crude worm extracts and in seemingly various oligomeric forms in the latter. The potential for oligomerization was supported by cross-linking experiments of recombinant OvDM9-1. Structure prediction suggested a ß-rich secondary structure of the protein and this was supported by a circular dichroism analysis. Molecular modeling in Phyre2 identified both MFP2 domains as distant homologs of OvDM9-1. The protein was located in tegumental type tissue and the cecal epithelium in the mature parasite. Recombinant OvDM9-1 was used as target in indirect ELISA but sera from infected hamsters showed only marginal reactivity towards it. It is proposed that OvDM9-1 and other members of this protein family have a role in cellular transport through functions on the cytoskeleton.

Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects.