Ichthyophthirius multifiliis is a highly pathogenic ciliate responsible for "white spot disease" in freshwater fish, causing significant economic losses to the global aquaculture industry. Early and accurate detection of this parasite is critical for disease control, however, current methods often require specialized equipment and trained personnel, limiting their applicability in resource-limited settings. In this study, a rapid, sensitive, and visual detection method for I. multifiliis was developed by combining enzymatic recombinase amplification (ERA) with lateral flow dipstick (LFD) technology. Specific primers and an internal probe targeting the mitochondrial cytochrome oxidase subunit I (COI) gene were designed. After optimizing the reaction conditions, the ERA-LFD assay achieved optimal performance at 43 ℃ for 18 min, with a detection limit of 100 copies/μL of recombinant plasmid DNA and the ability to detect as few as a single theront. The assay exhibited high specificity, showing no cross-reactivity with seven other common fish parasites (Argulus sp., Chilodonella uncinata, Cryptocaryon irritans, Trichodina sp., Gyrodactylus kobayashii, Trypanosoma sp., and Dactylogyrus sp.). Validation using experimentally infected goldfish (Carassius auratus) demonstrated 100% diagnostic concordance for both fish tissue and water samples. This ERA-LFD method combines isothermal amplification with visual readout, eliminating the need for thermal cyclers and specialized personnel. Its simplicity, speed, and high sensitivity make it a promising point-of-care testing (POCT) tool for early warning and surveillance of ichthyophthiriasis in aquaculture.
IgM detection is commonly employed for early diagnosis in human congenital toxoplasmosis. In small ruminants, accurate methods are needed to identify early foetal death (8-13 days post-infection), which often occurs without IgG seroconversion and parasite detection in the foetuses. The study aimed to develop and validate an ELISA based on lyophilized Toxoplasma gondii tachyzoites to detect anti-T. gondii IgM antibodies (IgM-ELISA) for sheep sera following WOAH guidelines. Using IgM-Western Blot (IgM-WB) as a reference test, sera panels comprising 735 sera from sheep experimentally- and naturally-infected with either T. gondii or other abortifacient pathogens were used. The IgM-TgSALUVET-ELISA demonstrated successful analytical validation, showing excellent precision (CV < 5%), good accuracy (R2 = 0.85-0.96) and analytical sensitivity (detection limit: DO = 0.1). While IgM-WB showed no cross-reactivity between T. gondii antigens and anti-Neospora caninum and anti-Chlamydia abortus antibodies, IgM ELISA yielded false positives. IgM kinetics analyses in T. gondii experimentally-infected sheep enabled early diagnosis capability of IgM-ELISA and IgM-WB, detecting IgM immunoglobulins at 10 and 13 dpi, aligning with the early abortion timeframe. The IgM-ELISA achieved 84% diagnostic sensitivity, 83.9% specificity, AUC = 0.88, and substantial agreement with IgM-WB (Kappa = 0.61). Field investigations based on paired and sequential IgM/IgG comparisons suggest the IgM detection is useful as a marker of recent T. gondii infection after excluding other abortifacient pathogens. However, prolonged anti-T. gondii IgM persistence may complicate the interpretation of results. In sum, the IgM-ELISA test showed good performance under the conditions tested, although potential interferences limit its use as a stand-alone assay. Therefore, a sequential diagnostic approach in the event of suspected foetal death during early T. gondii infection in a flock is proposed.
This study updates current information on the occurrence of Capillaria aerophila, Capillaria boehmi and Angiostrongylus vasorum infections in dogs from Italy, along with the evaluation of the occurrence of intestinal parasites. Fecal samples collected from a total of 2000 dogs living in the majority of the Italian regions were subjected to Baermann examination and flotation. Animals were selected according to a series of criteria to ensure the enrollment of dogs at risk to be infected by nematodes. A binomial logistic regression was performed to detect statistically significant associations between the positivity to Capillaria spp. and A. vasorum and selected epidemiological and clinical variables. Overall, 5.50%, 3.25% and 1.35% of dogs tested positive for C. aerophila, C. boehmi and A. vasorum respectively, with 1.30% of the animals being positive for at least two of them simultaneously. The statistical analysis showed significant associations between cardiorespiratory clinical signs and positivity to C. aerophila (p < 0.001; OR= 5.46; 95% CI: 2.04-14.66), C. boehmi (p < 0.001; OR= 10.38; 95% CI: 3.48-30.92), and A. vasorum (p < 0.001; OR= 17.81; 95% CI: 5.56-56.98). Significant statistical associations between the infection with C. aerophila (p < 0.001; OR= 3.92; 95% CI: 2.09-7.33) and with C. boehmi (p < 0.001; OR= 6.62; 95% CI: 2.64-16.55) and hunting dogs were also detected. Permanent outdoor housing was significantly associated with C. aerophila (p = 0.02; OR= 1.95; 95% CI: 1.14-3.35). Dogs ageing ≥ 5 years showed lower odds of positivity to both C. boehmi (p = 0.04; OR = 0.56; 95% CI: 0.33-0.95) and A. vasorum (p = 0.03; OR = 0.42; 95% CI: 0.18-0.94) than younger dogs. Mixed-breed status was also significantly associated with A. vasorum positivity (p = 0.03; OR = 3.13; 95% CI: 1.06-9.19). Ancylostomatidae (16.15%), Trichuris vulpis (12.45%) and ascarids (7.15%) were the most frequently detected intestinal parasites in the study population. Co-infections by respiratory and intestinal nematodes were found in 7.10% of the dogs, C. aerophila and Ancylostomatidae being the most frequent combination (1.80%). The present results confirm the epidemiological relevance of canine respiratory capillarioses and the established endemic occurrence of A. vasorum in Italy. The frequent detection of respiratory and intestinal nematodes, often in co-infections, emphasizes the importance of routine parasitological screening and of appropriate parasiticides in canine populations of Italy. This is particularly true in dogs who are more prone to be infected by endoparasites as those examined in the present study.
Amblyomma ovale is a widespread hard tick in Neotropics and it is an important vector of pathogens, particularly Rickettsia parkeri strain Atlantic rainforest, which causes a spotted fever illness in humans. Adult ticks primarily parasitize medium and large mammals, such as carnivores, while larvae and nymphs feed on small mammals and occasionally on birds. In Brazil, the species is found in all six major biomes (Amazon, Atlantic forest, Cerrado, Caatinga, Pantanal, Pampa), and is generally found in humid, forested areas. Despite its broad range, large-scale genetic studies have been lacking. This study analyzed the genetic diversity and population structure of A. ovale using five gene markers (four mitochondrial, one nuclear) in 57 specimens from Brazil (five biomes) plus Panama and Belize. High haplotype diversity was found for all markers, with numerous unique haplotypes and many biome-specific groupings. Phylogenetic analyses (maximum parsimony and maximum likelihood) revealed two major genetic clusters: one predominantly from Atlantic forest A region and another comprising samples from Atlantic forest B, other Brazilian biomes, and Central America. The nuclear marker (ITS2) exhibited low polymorphism and did not mirror mitochondrial patterns. This different pattern between mitochondrial and nuclear markers could be attributed to the over-resolution of mtDNA that can occur because the small inbreeding effective population size (Ne) of the mitochondrial genes may lead to coalescence of mtDNA lineages that are only temporarily isolated. Population genetic analyses (AMOVA and FST) revealed significant structuring among most regional populations, but no strong correlation between geographic and genetic distances (no isolation by distance). Neutrality tests indicated possible population expansion in the Atlantic forest B group. Overall, A. ovale exhibits high genetic diversity with some biome-linked clustering, especially between Atlantic forest subregions, as evidenced by mitochondrial markers. The tick’s broad host range and high movement may reduce isolation, thereby maintaining gene flow across regions. Given the public health importance of A. ovale as a vector of spotted fever, understanding this genetic variation could inform disease ecology and risk in different biomes.
Alveolar echinococcosis (AE), caused by the metacestode stage of Echinococcus multilocularis, is a highly lethal zoonosis. Its insidious progression frequently precludes diagnosis until advanced stages, and the paucity of effective early detection tools remains a critical impediment to disease control. In this study, we aimed to evaluate the diagnostic utility of EmCIP2A as a target antigen and develop monoclonal antibody-based tools for the serological and non-invasive detection of E. multilocularis infection. Six monoclonal antibodies (mAbs) were generated against EmCIP2A, a germinative cell-specific marker of the parasite. Isotyping identified 1F3C8 and 2F7G3 as IgG1, and 1G9E4, 3F1G4, 4B1C6, and 5A7C11 as IgG2b, all bearing kappa light chains. Western blotting and indirect immunofluorescence confirmed that all mAbs specifically recognize both recombinant and native EmCIP2A. Four linear B-cell epitopes were delineated with 1F3C8/5A7C11 targeted 229MADLSLTEER238, 4B1C6 bound 384HHSEATLNRL393, 2F7G3 recognized 432AEVEAKREAL441, and 1G9E4/3F1G4 reacted with 574TGNRVQPANA583. Based on epitope and affinity screening, a blocking ELISA employing HRP-1F3C8 was established (AUC = 0.8494; sensitivity, 90.63%; specificity, 72.73%). In addition, a double-antibody sandwich ELISA using 1F3C8 as capture antibody and HRP-2F7G3 as detection antibody was further developed, displaying excellent linearity (1-1000 ng/mL, R2 = 0.9973); preliminary fecal testing yielded detection rates of 70% in infected mice and 50% in infected dogs, confirming its utility for active infection surveillance across intermediate and definitive hosts. Collectively, the two ELISA systems provide complementary modalities, serological screening and non-invasive fecal antigen detection, which together expand the diagnostic repertoire against E. multilocularis infection.
