Pub Date : 2026-01-29DOI: 10.1016/j.vetpar.2026.110712
Francis Aduku , Jody L. Gookin , Jan S. Suchodolski , Lea J. Poellmann , Joe L. Luksovsky , Guilherme G. Verocai , Luis Fernando da Costa Medina , Mike Hong , Kathleen M. Aicher
Heterobilharzia americana is a trematode parasite that causes canine schistosomiasis, a disease capable of causing significant morbidity and mortality in dogs. Reliable diagnosis is essential for limiting disease progression. This study aimed to optimize and validate a previously developed TaqMan qPCR assay for detection of H. americana DNA in feces. The assay targets a highly repetitive non-coding DNA sequence located across multiple loci within the H. americana genome. PCR optimization involved gradient thermocycling and serial dilutions to refine annealing temperature and efficiency. PCR products were cloned and sequenced to confirm target specificity. Analytical sensitivity was assessed using serial two-fold dilutions of H. americana eggs spiked into feces and matrix-free samples, with PCR detection across replicates. Diagnostic sensitivity was assessed in 111 fecal samples from infected dogs using fecal sedimentation as gold standard. Analytical and diagnostic specificities were assessed by testing 54 fecal samples containing non-target parasites and 100 fecal samples from H. americana-uninfected dogs, respectively. Target specificity was confirmed by BLAST. Assay efficiency was 107 %. Matrix-dependent analytical sensitivity was 3 eggs/g feces (100 % replicates) and 1.5 eggs/g feces (60 % replicates); matrix-free sensitivity was 3 eggs/mL in saline. Diagnostic sensitivity was 98.2 % (95 % CI: 93.7–99.8 %). Both analytical and diagnostic specificities were 100 % (95 % CI: 93.6–100 % and 96.4–100 %, respectively). The assay demonstrated low intra-assay and inter-assay variability and minimal inter-operator variability across the dilution range tested. These findings support the optimized TaqMan qPCR assay as a highly sensitive and specific tool for detecting H. americana DNA in dog feces.
{"title":"Optimization and validation of a TaqMan real-time PCR for the detection of Heterobilharzia americana in dog feces","authors":"Francis Aduku , Jody L. Gookin , Jan S. Suchodolski , Lea J. Poellmann , Joe L. Luksovsky , Guilherme G. Verocai , Luis Fernando da Costa Medina , Mike Hong , Kathleen M. Aicher","doi":"10.1016/j.vetpar.2026.110712","DOIUrl":"10.1016/j.vetpar.2026.110712","url":null,"abstract":"<div><div><em>Heterobilharzia americana</em> is a trematode parasite that causes canine schistosomiasis, a disease capable of causing significant morbidity and mortality in dogs. Reliable diagnosis is essential for limiting disease progression. This study aimed to optimize and validate a previously developed TaqMan qPCR assay for detection of <em>H. americana</em> DNA in feces. The assay targets a highly repetitive non-coding DNA sequence located across multiple loci within the <em>H. americana</em> genome. PCR optimization involved gradient thermocycling and serial dilutions to refine annealing temperature and efficiency. PCR products were cloned and sequenced to confirm target specificity. Analytical sensitivity was assessed using serial two-fold dilutions of <em>H. americana</em> eggs spiked into feces and matrix-free samples, with PCR detection across replicates. Diagnostic sensitivity was assessed in 111 fecal samples from infected dogs using fecal sedimentation as gold standard. Analytical and diagnostic specificities were assessed by testing 54 fecal samples containing non-target parasites and 100 fecal samples from <em>H. americana</em>-uninfected dogs<em>,</em> respectively. Target specificity was confirmed by BLAST. Assay efficiency was 107 %. Matrix-dependent analytical sensitivity was 3 eggs/g feces (100 % replicates) and 1.5 eggs/g feces (60 % replicates); matrix-free sensitivity was 3 eggs/mL in saline. Diagnostic sensitivity was 98.2 % (95 % CI: 93.7–99.8 %). Both analytical and diagnostic specificities were 100 % (95 % CI: 93.6–100 % and 96.4–100 %, respectively). The assay demonstrated low intra-assay and inter-assay variability and minimal inter-operator variability across the dilution range tested. These findings support the optimized TaqMan qPCR assay as a highly sensitive and specific tool for detecting <em>H. americana</em> DNA in dog feces.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110712"},"PeriodicalIF":2.2,"publicationDate":"2026-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146114347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-29DOI: 10.1016/j.vetpar.2026.110711
Kyriánová Iveta Angela , Bieliková Lucia , Pytelková Jana , Pachulová Klára , Vacek Vojtěch , Fišerová Natálie , Vadlejch Jaroslav , Dvořák Jan
Haemonchus contortus, a highly pathogenic nematode of small ruminants, causes substantial economic losses worldwide. The transition from the infective third-stage larva (L3) to the metabolically active fourth-stage larva (L4) is critical for parasite survival, as L4 larvae initiate blood-feeding within the host. This study aimed to optimize in vitro cultivation conditions for L4 larvae and evaluate their metabolic activity, providing a basis for future research on parasite biology and anthelmintic development. We systematically tested six culture media (BME, DMEM, IMDM, RPMI 1640, M199, LB broth; LB without FBS) supplemented with antibiotics including gentamicin (significantly improved L4 development: 85 % vs 70 % without, p < 0.05) to determine optimal L3-to-L4 conditions. Larval survival was assessed by motility under inverted microscopy; L4 development by exsheathment, body thickening (600–800 μm), and genital primordium visibility. To assess metabolic status, we probed proteolytic activity, which represents a key function of blood-feeding larvae. A fluorogenic peptide substrate was employed to detect significant cysteine protease activity, serving as a marker of a competent digestive system. These findings establish a refined methodology for in vitro cultivation of L4 larvae and highlight their suitability for functional studies and drug screening. By optimizing culture conditions and demonstrating active proteolysis, this study provides a valuable platform for investigating stage-specific parasite biology and identifying novel therapeutic targets.
弯曲血蜱是一种小反刍动物的高致病性线虫，在世界范围内造成巨大的经济损失。从感染性第三期幼虫（L3）到代谢活跃的第四期幼虫（L4）的过渡对寄生虫的生存至关重要，因为L4期幼虫开始在宿主内吸血。本研究旨在优化L4幼虫体外培养条件，评价其代谢活性，为今后寄生虫生物学和驱虫发育研究提供依据。我们系统地测试了6种培养基（BME、DMEM、IMDM、RPMI 1640、M199、LB肉汤、LB不含FBS）添加庆大霉素等抗生素（显著改善L4发育：85 % vs 70 %，p <； 0.05），以确定l3 -L4的最佳条件。倒置显微镜下通过运动评估幼虫存活率；L4的发育通过呼鞘、体增厚（600-800 μm）和生殖器原基可见性。为了评估代谢状态，我们研究了蛋白质水解活性，这是食血幼虫的一个关键功能。荧光肽底物被用来检测显著的半胱氨酸蛋白酶活性，作为消化系统能力的标志。这些发现为L4幼虫的体外培养建立了一种完善的方法，并强调了它们在功能研究和药物筛选方面的适用性。通过优化培养条件和证明活性蛋白水解，本研究为研究寄生虫的阶段特异性生物学和确定新的治疗靶点提供了一个有价值的平台。
{"title":"Optimizing in vitro cultivation of Haemonchus contortus larvae: Comparative assessment of growth conditions","authors":"Kyriánová Iveta Angela , Bieliková Lucia , Pytelková Jana , Pachulová Klára , Vacek Vojtěch , Fišerová Natálie , Vadlejch Jaroslav , Dvořák Jan","doi":"10.1016/j.vetpar.2026.110711","DOIUrl":"10.1016/j.vetpar.2026.110711","url":null,"abstract":"<div><div><em>Haemonchus contortus,</em> a highly pathogenic nematode of small ruminants, causes substantial economic losses worldwide. The transition from the infective third-stage larva (L<sub>3</sub>) to the metabolically active fourth-stage larva (L<sub>4</sub>) is critical for parasite survival, as L<sub>4</sub> larvae initiate blood-feeding within the host. This study aimed to optimize <em>in vitro</em> cultivation conditions for L<sub>4</sub> larvae and evaluate their metabolic activity, providing a basis for future research on parasite biology and anthelmintic development. We systematically tested six culture media (BME, DMEM, IMDM, RPMI 1640, M199, LB broth; LB without FBS) supplemented with antibiotics including gentamicin (significantly improved L<sub>4</sub> development: 85 % vs 70 % without, p < 0.05) to determine optimal L<sub>3</sub>-to-L<sub>4</sub> conditions. Larval survival was assessed by motility under inverted microscopy; L<sub>4</sub> development by exsheathment, body thickening (600–800 μm), and genital primordium visibility. To assess metabolic status, we probed proteolytic activity, which represents a key function of blood-feeding larvae. A fluorogenic peptide substrate was employed to detect significant cysteine protease activity, serving as a marker of a competent digestive system. These findings establish a refined methodology for <em>in vitro</em> cultivation of L<sub>4</sub> larvae and highlight their suitability for functional studies and drug screening. By optimizing culture conditions and demonstrating active proteolysis, this study provides a valuable platform for investigating stage-specific parasite biology and identifying novel therapeutic targets.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110711"},"PeriodicalIF":2.2,"publicationDate":"2026-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146079216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-28DOI: 10.1016/j.vetpar.2026.110708
Ana Carolina de Souza Chagas, Gustavo Avelar Sousa, Rafaela Tami Ikeda Kapritchkoff, Luís Adriano Anholeto, Leonardo Aparecido Lima Dos Santos, Vera Lúcia de Castro, Waldomiro Barion-Júnior, Alessandro Pelegrine Minho
Amblyomma sculptum is the main vector of Rickettsia rickettsii, the etiological agent of Brazilian spotted fever (BSF), a relevant zoonosis due to the increasing number of human deaths. This study evaluated the efficacy of seven commercial products against A. sculptum larvae, with comparative assays also conducted for Rhipicephalus microplus. Formulations were first evaluated using the larval packet test (LPT), then in semi-field trials in Urochloa (Syn. Brachiaria) brizantha, Panicum maximum, and Cynodon spp. forages, and last in an ecotoxicity assay. In the LPT, LC99 values for the products ranged from 0.0024 to 1.39 mg/mL for A. sculptum, and from 0.0093 to 0.56 mg/mL for R. microplus. The efficacy rates against A. sculptum in Urochloa were 93.1 %, 96.4 %, and 97.0 % for lambda-cyhalothrin, thiamethoxam + lambda-cyhalothrin, and abamectin, respectively. For R. microplus, the efficacies were 80.8 %, 95.1 % and 75.2 % for lambda-cyhalothrin, 89.8 %, 97.9 % and 88.8 % for thiamethoxam + lambda-cyhalothrin, and 40.7 %, 56.0 % and 6.2 % for abamectin, in Urochloa, Cynodon, and Panicum, respectively. Climatic conditions were similar across forages, favouring larval survival, except for the effect of rainfall on A. sculptum. In ecotoxicity assays, lambda-cyhalothrin and thiamethoxam + lambda-cyhalothrin had mild effects on recovery, growth and fertility of Caenorhabditis elegans. Thiamethoxam + lambda-cyhalothrin offers short-term larval control under semi-field conditions and is another option for managing A. sculptum in areas at risk of BSF. Further studies are required to assess efficacy against nymphs and adult ticks, environmental impacts, effects on non-target organisms, and residual activity, to ensure the sustainable use of these acaricides.
{"title":"Exploring new acaricidal compounds for environmental control of Amblyomma sculptum (Brazilian spotted fever agent): An in vitro and semi-field comparative study with Rhipicephalus microplus.","authors":"Ana Carolina de Souza Chagas, Gustavo Avelar Sousa, Rafaela Tami Ikeda Kapritchkoff, Luís Adriano Anholeto, Leonardo Aparecido Lima Dos Santos, Vera Lúcia de Castro, Waldomiro Barion-Júnior, Alessandro Pelegrine Minho","doi":"10.1016/j.vetpar.2026.110708","DOIUrl":"https://doi.org/10.1016/j.vetpar.2026.110708","url":null,"abstract":"<p><p>Amblyomma sculptum is the main vector of Rickettsia rickettsii, the etiological agent of Brazilian spotted fever (BSF), a relevant zoonosis due to the increasing number of human deaths. This study evaluated the efficacy of seven commercial products against A. sculptum larvae, with comparative assays also conducted for Rhipicephalus microplus. Formulations were first evaluated using the larval packet test (LPT), then in semi-field trials in Urochloa (Syn. Brachiaria) brizantha, Panicum maximum, and Cynodon spp. forages, and last in an ecotoxicity assay. In the LPT, LC<sub>99</sub> values for the products ranged from 0.0024 to 1.39 mg/mL for A. sculptum, and from 0.0093 to 0.56 mg/mL for R. microplus. The efficacy rates against A. sculptum in Urochloa were 93.1 %, 96.4 %, and 97.0 % for lambda-cyhalothrin, thiamethoxam + lambda-cyhalothrin, and abamectin, respectively. For R. microplus, the efficacies were 80.8 %, 95.1 % and 75.2 % for lambda-cyhalothrin, 89.8 %, 97.9 % and 88.8 % for thiamethoxam + lambda-cyhalothrin, and 40.7 %, 56.0 % and 6.2 % for abamectin, in Urochloa, Cynodon, and Panicum, respectively. Climatic conditions were similar across forages, favouring larval survival, except for the effect of rainfall on A. sculptum. In ecotoxicity assays, lambda-cyhalothrin and thiamethoxam + lambda-cyhalothrin had mild effects on recovery, growth and fertility of Caenorhabditis elegans. Thiamethoxam + lambda-cyhalothrin offers short-term larval control under semi-field conditions and is another option for managing A. sculptum in areas at risk of BSF. Further studies are required to assess efficacy against nymphs and adult ticks, environmental impacts, effects on non-target organisms, and residual activity, to ensure the sustainable use of these acaricides.</p>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"110708"},"PeriodicalIF":2.2,"publicationDate":"2026-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146259483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.vetpar.2026.110705
Maria Vitória Lamóglia Bastos Ferreira , Edna Barcelos Alves , Caroline Bittencourt Miranda , Vanessa Cola Thomazini , Ricardo Augusto Mendonça Vieira , Luis Fonseca Matos , Clóvis de Paula Santos
Gastrointestinal nematodes pose a major threat to livestock health and productivity, and the growing inefficacy of commercial anthelmintics highlights the need for alternative control methods. This study evaluated BioVerm®, a Brazilian commercial product (BCP) based on the nematophagous fungus Arthrobotrys flagrans, using three criteria: chlamydospore count per gram (CCG), predatory activity, and efficacy in reducing infective larvae (L3). CCG was quantified using a Neubauer chamber. Predatory activity was assessed by inoculating Panagrellus spp. in Petri dishes. Efficacy was evaluated through coprocultures treated or not with (BCP), including post-gastrointestinal tract analysis using sheep feces. Three commercial batches were tested. The mean CCG ranged from 8333 to 12,500, significantly below the 5 × 10⁵ spores per gram stated on the label. Fungal growth was observed in only one batch, and high contamination was found in another. No significant reduction in L3 counts was detected in either in vitro or in vivo assays. These results indicate low fungal viability and support the urgent need for quality control measures to ensure BCP efficacy.
{"title":"Evaluation of Arthrobotrys flagrans concentration, predatory activity, and efficacy in a commercial product","authors":"Maria Vitória Lamóglia Bastos Ferreira , Edna Barcelos Alves , Caroline Bittencourt Miranda , Vanessa Cola Thomazini , Ricardo Augusto Mendonça Vieira , Luis Fonseca Matos , Clóvis de Paula Santos","doi":"10.1016/j.vetpar.2026.110705","DOIUrl":"10.1016/j.vetpar.2026.110705","url":null,"abstract":"<div><div>Gastrointestinal nematodes pose a major threat to livestock health and productivity, and the growing inefficacy of commercial anthelmintics highlights the need for alternative control methods. This study evaluated BioVerm®, a Brazilian commercial product (BCP) based on the nematophagous fungus <em>Arthrobotrys flagrans</em>, using three criteria: chlamydospore count per gram (CCG), predatory activity, and efficacy in reducing infective larvae (L3). CCG was quantified using a Neubauer chamber. Predatory activity was assessed by inoculating <em>Panagrellus</em> spp. in Petri dishes. Efficacy was evaluated through coprocultures treated or not with (BCP), including post-gastrointestinal tract analysis using sheep feces. Three commercial batches were tested. The mean CCG ranged from 8333 to 12,500, significantly below the 5 × 10⁵ spores per gram stated on the label. Fungal growth was observed in only one batch, and high contamination was found in another. No significant reduction in L3 counts was detected in either <em>in vitro</em> or <em>in vivo</em> assays. These results indicate low fungal viability and support the urgent need for quality control measures to ensure BCP efficacy.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110705"},"PeriodicalIF":2.2,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146079215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.vetpar.2026.110707
Anna Sophia Feix , Maibritt Mardahl , Johan Palmfeldt , Rikke Brødsgaard Kjærup , Tina Sørensen Dalgaard
Ascaridia galli is one of the most prevalent intestinal nematodes of domesticated chickens and the primary cause of avian ascaridiasis, a globally important disease in poultry production. Infection rates often reach 70–90 % in free-range systems due to its direct life cycle and efficient environmental transmission. Despite its impact, the molecular mechanisms governing A. galli host-parasite interactions remain poorly understood. A. galli belongs to Clade III nematodes, which include several species of major veterinary importance. Comparative genomic studies Clade III-nematodes have revealed expansions in gene families associated with key parasitic traits such as immune modulation, tissue migration, and adaptation to host environments. Proteomics provides a powerful approach for characterizing these processes by identifying proteins essential for development, metabolism, reproduction, and host interaction. However, current knowledge is based almost exclusively on mammalian-infecting species, and no developmental proteome has previously been mapped for A. galli. By profiling protein expression across life stages, this study produces the first proteomic dataset for A. galli. In total, 852 worm proteins were identified. Of these, 452 proteins were shared across female adults, male adults, and larvae, while each group also displayed distinct protein sets. Male worms shared a substantial subset of 327 proteins with females but not larvae, while larvae and females shared 18 proteins. These findings demonstrate that protein expression in parasitic nematodes is strongly sex-specific, with males typically expressing proteins linked to spermatogenesis and motility, and females expressing vitellogenins and lipid-binding proteins.
{"title":"Proteomic profiling of Ascaridia galli reveals sex- and stage-specific protein signatures","authors":"Anna Sophia Feix , Maibritt Mardahl , Johan Palmfeldt , Rikke Brødsgaard Kjærup , Tina Sørensen Dalgaard","doi":"10.1016/j.vetpar.2026.110707","DOIUrl":"10.1016/j.vetpar.2026.110707","url":null,"abstract":"<div><div><em>Ascaridia galli</em> is one of the most prevalent intestinal nematodes of domesticated chickens and the primary cause of avian ascaridiasis, a globally important disease in poultry production. Infection rates often reach 70–90 % in free-range systems due to its direct life cycle and efficient environmental transmission. Despite its impact, the molecular mechanisms governing <em>A. galli</em> host-parasite interactions remain poorly understood. <em>A. galli</em> belongs to Clade III nematodes, which include several species of major veterinary importance. Comparative genomic studies Clade III-nematodes have revealed expansions in gene families associated with key parasitic traits such as immune modulation, tissue migration, and adaptation to host environments. Proteomics provides a powerful approach for characterizing these processes by identifying proteins essential for development, metabolism, reproduction, and host interaction. However, current knowledge is based almost exclusively on mammalian-infecting species, and no developmental proteome has previously been mapped for <em>A. galli.</em> By profiling protein expression across life stages, this study produces the first proteomic dataset for <em>A. galli</em>. In total, 852 worm proteins were identified. Of these, 452 proteins were shared across female adults, male adults, and larvae, while each group also displayed distinct protein sets. Male worms shared a substantial subset of 327 proteins with females but not larvae, while larvae and females shared 18 proteins. These findings demonstrate that protein expression in parasitic nematodes is strongly sex-specific, with males typically expressing proteins linked to spermatogenesis and motility, and females expressing vitellogenins and lipid-binding proteins.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110707"},"PeriodicalIF":2.2,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039163","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-19DOI: 10.1016/j.vetpar.2026.110704
Connor Leong , Ruby Scanlon , Aisling Kyne , Thomas J. Sharpton , Michael L. Kent
Zebrafish (Danio rerio) are a widely used biomedical model and offers powerful high-throughput screening capabilities for assessing chemical bioactivity. We have previously employed adult zebrafish infected with the intestinal nematode Pseudocapillaria tomentosa to investigate nematode–microbiome interactions, nematode-promoted intestinal neoplasia, and anthelmintic drug discovery. Here we transition this model to a larval zebrafish infection infection to enable larger-scale experimentation and ultimately accelerate anthelmintic discovery. Infection conditions were optimized across 5–30 days post fertilization (dpf). The 30 dpf larvae exhibited the most robust and reproducible infections in multi-well formats, as well as the highest survival relative to younger stages. We described worm development from hatching through larval progression and maturation, addressing a major gap in foundational data with fish capillarids. Using in vitro–hatched larvae and infected larval and adult zebrafish, we documented developmental trajectories from 1 to 37 days post-exposure. Change-point analysis identified putative ecdysis transitions at the following worm lengths (mm): L1/L2 = 0.220, L2/L3 = 0.571, L3/L4 = 1.174, and L4/L5 = 1.584. Finally, we demonstrated proof-of-concept for anthelmintic screening by exposing fish to larvated eggs in the presence of emamectin benzoate (macrocyclic lactone) or fenbendazole (benzimidazole). Both compounds reduced worm burdens after 3 days, with the strongest effects at higher concentrations (0.7 µM emamectin benzoate; 0.3 µM fenbendazole). Together, these findings establishes a proof of concept for larval zebrafish infection platform which bridges the gap between in vitro and mammalian in vivo assays, enabling scalable, efficient, and biologically relevant screening for anthelmintic drug discovery.
{"title":"Pseudocapillaria tomentosa infections in laboratory larval and Adult Zebrafish (Danio rerio): Development and advances in an in vivo anthelmintic drug discovery model","authors":"Connor Leong , Ruby Scanlon , Aisling Kyne , Thomas J. Sharpton , Michael L. Kent","doi":"10.1016/j.vetpar.2026.110704","DOIUrl":"10.1016/j.vetpar.2026.110704","url":null,"abstract":"<div><div>Zebrafish (<em>Danio rerio</em>) are a widely used biomedical model and offers powerful high-throughput screening capabilities for assessing chemical bioactivity. We have previously employed adult zebrafish infected with the intestinal nematode <em>Pseudocapillaria tomentosa</em> to investigate nematode–microbiome interactions, nematode-promoted intestinal neoplasia, and anthelmintic drug discovery. Here we transition this model to a larval zebrafish infection infection to enable larger-scale experimentation and ultimately accelerate anthelmintic discovery. Infection conditions were optimized across 5–30 days post fertilization (dpf). The 30 dpf larvae exhibited the most robust and reproducible infections in multi-well formats, as well as the highest survival relative to younger stages. We described worm development from hatching through larval progression and maturation, addressing a major gap in foundational data with fish capillarids. Using <em>in vitro</em>–hatched larvae and infected larval and adult zebrafish, we documented developmental trajectories from 1 to 37 days post-exposure. Change-point analysis identified putative ecdysis transitions at the following worm lengths (mm): L1/L2 = 0.220, L2/L3 = 0.571, L3/L4 = 1.174, and L4/L5 = 1.584. Finally, we demonstrated proof-of-concept for anthelmintic screening by exposing fish to larvated eggs in the presence of emamectin benzoate (macrocyclic lactone) or fenbendazole (benzimidazole). Both compounds reduced worm burdens after 3 days, with the strongest effects at higher concentrations (0.7 µM emamectin benzoate; 0.3 µM fenbendazole). Together, these findings establishes a proof of concept for larval zebrafish infection platform which bridges the gap between in vitro and mammalian in vivo assays, enabling scalable, efficient, and biologically relevant screening for anthelmintic drug discovery.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110704"},"PeriodicalIF":2.2,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146100693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-19DOI: 10.1016/j.vetpar.2026.110703
Xueqiu Chen , Lingyun Mou , Manqin Deng , Jingju Zhang , Jiaming Yu , Jianfang Li , Hongli Zhang , Guangxu Ma , Yi Yang , Aifang Du
Haemonchosis, caused by the parasitic nematode Haemonchus contortus, poses a significant global threat to small ruminant health and productivity. Current diagnostic methods, such as fecal egg flotation, cannot detect early-stage or latent infections, hindering timely intervention. Building on prior identification of the secreted BPTI/Kunitz inhibitor domain-containing protein SPI-I8 as a key H. contortus antigen released during initial infection, we developed two diagnostic methods using recombinant Hc-SPI-I8B (rHc-SPI-I8B). First, an indirect ELISA with rHc-SPI-I8B as the coating antigen demonstrated detection of H. contortus infection in sheep as early as 10 days post-infection (dpi), with a sensitivity of 1:200. Concurrently, an immunochromatographic test (ICT) was established by conjugating rHc-SPI-I8B with 20 nm gold particles, achieving a sensitivity of 1:40, detecting antibodies at 11 dpi, and remaining effective beyond 100 dpi. Both methods exhibited high specificity, showing no cross-reactivity with positive sera of Echinococcus granulosus, Fasciola hepatica, or Fasciola gigantica. Additionally, they showed high stability and reproducibility (inter-/intra-assay CV <10 % for ELISA). Both approaches provide efficient tools for early diagnosis of haemonchosis.
{"title":"Development of an indirect ELISA and an immunochromatographic test method for early detection of Haemonchus contortus infection in sheep","authors":"Xueqiu Chen , Lingyun Mou , Manqin Deng , Jingju Zhang , Jiaming Yu , Jianfang Li , Hongli Zhang , Guangxu Ma , Yi Yang , Aifang Du","doi":"10.1016/j.vetpar.2026.110703","DOIUrl":"10.1016/j.vetpar.2026.110703","url":null,"abstract":"<div><div>Haemonchosis, caused by the parasitic nematode <em>Haemonchus contortus</em>, poses a significant global threat to small ruminant health and productivity. Current diagnostic methods, such as fecal egg flotation, cannot detect early-stage or latent infections, hindering timely intervention. Building on prior identification of the secreted BPTI/Kunitz inhibitor domain-containing protein SPI-I8 as a key <em>H. contortus</em> antigen released during initial infection, we developed two diagnostic methods using recombinant <em>Hc</em>-SPI-I8B (r<em>Hc</em>-SPI-I8B). First, an indirect ELISA with r<em>Hc</em>-SPI-I8B as the coating antigen demonstrated detection of <em>H. contortus</em> infection in sheep as early as 10 days post-infection (dpi), with a sensitivity of 1:200. Concurrently, an immunochromatographic test (ICT) was established by conjugating r<em>Hc</em>-SPI-I8B with 20 nm gold particles, achieving a sensitivity of 1:40, detecting antibodies at 11 dpi, and remaining effective beyond 100 dpi. Both methods exhibited high specificity, showing no cross-reactivity with positive sera of <em>Echinococcus granulosus</em>, <em>Fasciola hepatica</em>, or <em>Fasciola gigantica</em>. Additionally, they showed high stability and reproducibility (inter-/intra-assay CV <10 % for ELISA). Both approaches provide efficient tools for early diagnosis of haemonchosis.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110703"},"PeriodicalIF":2.2,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-18DOI: 10.1016/j.vetpar.2026.110702
Sara Marcos-Herraiz , Sara Alonso Fernández , María José Irisarri , Jaime Arroyo Díaz , Francisco Ponce-Gordo , Azucena González-Coloma , Juliana Navarro Rocha , Iris Azami-Conesa , María Teresa Gómez-Muñoz , María Bailén
Lamiaceae and Asteraceae plant species have been widely used in Mediterranean ethnomedicine for gastrointestinal disorders. They are also known for their antioxidant, anti-inflammatory, anti-bacterial, anti-parasite, and anti-virus properties. Giardia duodenalis is the most prevalent intestinal protozoon in children and young dogs worldwide. Its zoonotic potential and frequent therapeutic failures with nitroimidazoles underscore the urgent need for alternative treatments. This study investigated the antigiardial activity of essential oils (EOs) from 22 medicinal plants belonging to Lamiaceae and Asteraceae, together with their major constituents. EO composition was determined by a metabolomic approach (GC-MS). Parasite metabolic activity was assessed using the MTT assay, and ultrastructural changes were examined by Transmission Electron Microscopy. The strongest antigiardial effects were observed with Lavandula luisieri, Thymus vulgaris, Mentha suaveolens, Satureja montana (IC50 <25), L. lanata, and T. zygis, (IC50= 27.9–71.5 µg/ml). The highest selective indexes were obtained with γ-terpinene, caryophyllene oxide, carvacrol and thymol (SI≥1.3–2.4). Synergistic interactions were detected with linalyl acetate and linalool (present in Lavandula EOs), linalyl acetate with ρ-cymene or thymol, or combinations of ρ-cymene, γ-terpinene, thymol, and carvacrol (present in Satureja EOs). Transmission Electron Microscopy revealed membranolysis, enlarged periplasmic vacuoles, and cytoplasmic loss in trophozoites exposed to γ-terpinene after 1 h. These findings provide phytotherapeutic evidence supporting essential oils from Lavandula, Mentha, Thymus, and Satureja as promising antigiardial agents. Their main components γ-terpinene, caryophyllene oxide, carvacrol and thymol could have potential applications in veterinary parasitology.
{"title":"Phytotherapeutic potential of Lamiaceae essential oils and their monoterpenes against Giardia duodenalis.","authors":"Sara Marcos-Herraiz , Sara Alonso Fernández , María José Irisarri , Jaime Arroyo Díaz , Francisco Ponce-Gordo , Azucena González-Coloma , Juliana Navarro Rocha , Iris Azami-Conesa , María Teresa Gómez-Muñoz , María Bailén","doi":"10.1016/j.vetpar.2026.110702","DOIUrl":"10.1016/j.vetpar.2026.110702","url":null,"abstract":"<div><div>Lamiaceae and Asteraceae plant species have been widely used in Mediterranean ethnomedicine for gastrointestinal disorders. They are also known for their antioxidant, anti-inflammatory, anti-bacterial, anti-parasite, and anti-virus properties. <em>Giardia duodenalis</em> is the most prevalent intestinal protozoon in children and young dogs worldwide. Its zoonotic potential and frequent therapeutic failures with nitroimidazoles underscore the urgent need for alternative treatments. This study investigated the antigiardial activity of essential oils (EOs) from 22 medicinal plants belonging to Lamiaceae and Asteraceae, together with their major constituents. EO composition was determined by a metabolomic approach (GC-MS). Parasite metabolic activity was assessed using the MTT assay, and ultrastructural changes were examined by Transmission Electron Microscopy. The strongest antigiardial effects were observed with <em>Lavandula luisieri</em>, <em>Thymus vulgaris, Mentha suaveolens, Satureja montana</em> (IC<sub>50</sub> <25), <em>L. lanata</em>, and <em>T. zygis,</em> (IC<sub>50</sub>= 27.9–71.5 µg/ml). The highest selective indexes were obtained with γ-terpinene, caryophyllene oxide, carvacrol and thymol (SI≥1.3–2.4). Synergistic interactions were detected with linalyl acetate and linalool (present in <em>Lavandula</em> EOs), linalyl acetate with ρ-cymene or thymol, or combinations of ρ-cymene, γ-terpinene, thymol, and carvacrol (present in <em>Satureja</em> EOs). Transmission Electron Microscopy revealed membranolysis, enlarged periplasmic vacuoles, and cytoplasmic loss in trophozoites exposed to γ-terpinene after 1 h. These findings provide phytotherapeutic evidence supporting essential oils from <em>Lavandula</em>, <em>Mentha</em>, <em>Thymus,</em> and <em>Satureja</em> as promising antigiardial agents. Their main components γ-terpinene, caryophyllene oxide, carvacrol and thymol could have potential applications in veterinary parasitology.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110702"},"PeriodicalIF":2.2,"publicationDate":"2026-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-13DOI: 10.1016/j.vetpar.2026.110700
Li-Qun Wang , Guang-Xue Liu , Pan-Hong Liang , Li Li , Tao-Shan Li , Ai-Ming Guo , Tharheer Oluwashola Amuda , Ke-Ke Wu , Yi-Xuan Wu , Hong Yin , Hong-Bin Yan , Xue-Nong Luo
Cysticercosis, caused by the larval stage of T. solium, remains a major neglected tropical disease with severe clinical and socioeconomic consequences in endemic regions. Although cyclic AMP-dependent protein kinase (PKA) signaling is fundamental to parasite development and survival, its therapeutic relevance in T. solium has not been comprehensively explored. This study provides an integrated molecular and functional characterization of the T. solium PKA catalytic subunit (TsPKA-c) and evaluates its potential as a novel therapeutic target for cysticercosis control. Recombinant TsPKA-c was successfully expressed in Pichia pastoris and purified in an active form, demonstrating robust kinase activity. Phylogenetic analysis revealed strong evolutionary conservation among cestode PKA homologues, underscoring its essential biological function. Immunohistochemical analysis confirmed stage-specific expression in both larval and adult parasite forms. Optimal enzymatic activity occurred at pH 7.5 and 35°C, with kinetic parameters showing a Km of 36.37 ± 2.64 µM and Vmax of 0.7648 ± 0.013 nmol/min/µg. Pharmacological inhibition experiments identified H89 as a highly potent inhibitor of TsPKA-c (IC50 = 14.55 ± 1.5 µM), exhibiting greater efficacy than PKI (14−22) (IC50 = 23.09 ± 0.5 µM). Both inhibitors induced significant dose- and time-dependent mortality in T. pisiformis cysticerci, with H89 causing rapid lethality. Metabolic analyses demonstrated a marked reduction in glucose uptake following TsPKA-c inhibition, accompanied by alterations in excretory-secretory protein profiles. Collectively, these findings establish TsPKA-c as a critical regulator of parasite metabolism and survival, supporting its strong candidacy as a promising molecular target for the development of novel anti-cysticercosis therapeutic strategies.
{"title":"The catalytic subunit of cAMP-dependent protein kinase in Taenia solium cysticerci (TsPKA-c): A promising molecular target for the development of novel anti-cysticercosis therapeutics","authors":"Li-Qun Wang , Guang-Xue Liu , Pan-Hong Liang , Li Li , Tao-Shan Li , Ai-Ming Guo , Tharheer Oluwashola Amuda , Ke-Ke Wu , Yi-Xuan Wu , Hong Yin , Hong-Bin Yan , Xue-Nong Luo","doi":"10.1016/j.vetpar.2026.110700","DOIUrl":"10.1016/j.vetpar.2026.110700","url":null,"abstract":"<div><div>Cysticercosis, caused by the larval stage of <em>T. solium</em>, remains a major neglected tropical disease with severe clinical and socioeconomic consequences in endemic regions. Although cyclic AMP-dependent protein kinase (PKA) signaling is fundamental to parasite development and survival, its therapeutic relevance in <em>T. solium</em> has not been comprehensively explored. This study provides an integrated molecular and functional characterization of the <em>T. solium</em> PKA catalytic subunit (TsPKA-c) and evaluates its potential as a novel therapeutic target for cysticercosis control. Recombinant TsPKA-c was successfully expressed in <em>Pichia pastoris</em> and purified in an active form, demonstrating robust kinase activity. Phylogenetic analysis revealed strong evolutionary conservation among cestode PKA homologues, underscoring its essential biological function. Immunohistochemical analysis confirmed stage-specific expression in both larval and adult parasite forms. Optimal enzymatic activity occurred at pH 7.5 and 35°C, with kinetic parameters showing a Km of 36.37 ± 2.64 µM and Vmax of 0.7648 ± 0.013 nmol/min/µg. Pharmacological inhibition experiments identified H89 as a highly potent inhibitor of TsPKA-c (IC50 = 14.55 ± 1.5 µM), exhibiting greater efficacy than PKI (14−22) (IC50 = 23.09 ± 0.5 µM). Both inhibitors induced significant dose- and time-dependent mortality in <em>T. pisiformis</em> cysticerci, with H89 causing rapid lethality. Metabolic analyses demonstrated a marked reduction in glucose uptake following TsPKA-c inhibition, accompanied by alterations in excretory-secretory protein profiles. Collectively, these findings establish TsPKA-c as a critical regulator of parasite metabolism and survival, supporting its strong candidacy as a promising molecular target for the development of novel anti-cysticercosis therapeutic strategies.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110700"},"PeriodicalIF":2.2,"publicationDate":"2026-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145980410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-12DOI: 10.1016/j.vetpar.2026.110701
Olfat A. Mahdy , Mai A. Salem , Mohamed S. Kamel , Mohamed A. El-Saied , Asmaa K. Al-Mokaddem , Radwa Ashour , Reem M. Ramadan
Ovine fascioliasis, caused by Fasciola species, challenges livestock productivity worldwide and remains insufficiently characterized at the molecular and immunological levels in endemic regions, such as Egypt. In 150 slaughtered sheep, the prevalence was determined, mitochondrial COX1 was sequenced for species identity, hepatic cytokine transcripts were quantified via qRT-PCR, oxidative stress biomarkers (MDA, TAC, TOS, and OSI) were assayed, and histopathology was evaluated. The prevalence was 15.3 %, with non-significant sex/season effects. COX1 phylogenetics confirmed that Fasciola hepatica identity was closely related to global haplotypes. Infected livers showed upregulation of pro-inflammatory (IL-1β) 4.0 × (, TNF-α) 6.6 × () and regulatory (IL-10 (5.3 ×), TGF-β (4.7 ×), and IL-4 (4.4 ×) cytokines, with downregulated IFN-γ (0.45 ×). Oxidative stress markers, including MDA (5 ×), TOS (6.1 ×), and OSI (3.6 ×), were significantly elevated, alongside a compensatory rise in TAC (7.8 ×). Histological examination revealed hepatocellular degeneration, bile duct hyperplasia, inflammatory infiltration, and fibrosis. These results elucidate the complex immune modulation and oxidative imbalance during F. hepatica infection and emphasize the necessity of integrated molecular and functional diagnostics to enhance fascioliasis management in sheep.
{"title":"Concurrent evaluation of oxidative stress biomarkers, cytokine expression, molecular identification, and histopathological findings in sheep naturally infected with Fasciola hepatica","authors":"Olfat A. Mahdy , Mai A. Salem , Mohamed S. Kamel , Mohamed A. El-Saied , Asmaa K. Al-Mokaddem , Radwa Ashour , Reem M. Ramadan","doi":"10.1016/j.vetpar.2026.110701","DOIUrl":"10.1016/j.vetpar.2026.110701","url":null,"abstract":"<div><div>Ovine fascioliasis, caused by <em>Fasciola</em> species, challenges livestock productivity worldwide and remains insufficiently characterized at the molecular and immunological levels in endemic regions, such as Egypt. In 150 slaughtered sheep, the prevalence was determined, mitochondrial COX1 was sequenced for species identity, hepatic cytokine transcripts were quantified via qRT-PCR, oxidative stress biomarkers (MDA, TAC, TOS, and OSI) were assayed, and histopathology was evaluated. The prevalence was 15.3 %, with non-significant sex/season effects. COX1 phylogenetics confirmed that <em>Fasciola hepatica</em> identity was closely related to global haplotypes. Infected livers showed upregulation of pro-inflammatory (IL-1β) 4.0 × (, TNF-α) 6.6 × () and regulatory (IL-10 (5.3 ×), TGF-β (4.7 ×), and IL-4 (4.4 ×) cytokines, with downregulated IFN-γ (0.45 ×). Oxidative stress markers, including MDA (5 ×), TOS (6.1 ×), and OSI (3.6 ×), were significantly elevated, alongside a compensatory rise in TAC (7.8 ×). Histological examination revealed hepatocellular degeneration, bile duct hyperplasia, inflammatory infiltration, and fibrosis. These results elucidate the complex immune modulation and oxidative imbalance during <em>F. hepatica</em> infection and emphasize the necessity of integrated molecular and functional diagnostics to enhance fascioliasis management in sheep.</div></div>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"343 ","pages":"Article 110701"},"PeriodicalIF":2.2,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145980408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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