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.
Protozoan parasites of the genus Eimeria represent important enteric pathogens of South American camelids (SACs), particularly affecting juvenile alpacas and llamas. Despite their veterinary and economic significance, current knowledge regarding camelid coccidiosis remains limited, especially in relation to species taxonomy, endogenous development, molecular diagnostics, and species-specific treatment strategies. This review summarizes current knowledge on Eimeria species infecting SACs, including their morphology, life cycle, epidemiology, pathogenicity, diagnosis, treatment, and prevention. Particular attention is given to the unique biological characteristics of large camelid coccidia such as Eimeria macusaniensis, diagnostic challenges associated with prolonged prepatent periods and intermittent oocyst shedding, and the limited availability of validated molecular diagnostic methods. The review further highlights the lack of approved anticoccidial therapies and the frequent reliance on treatment protocols extrapolated from domestic ruminants. Finally, major research gaps and future perspectives are discussed, emphasizing the need for improved molecular characterization, early diagnostic tools, and evidence-based therapeutic approaches for camelid coccidiosis.
Accurate tick species identification is essential for vector surveillance and control of tick-borne diseases, but conventional morphological diagnosis remains challenging for closely related Hyalomma species. This study developed and evaluated a deep learning-based framework for automated recognition of five medically and veterinary important Hyalomma species from Tunisia. A dataset of 1344 dorsal and ventral images of morphologically identified ticks was generated from livestock specimens, including 484 H. marginatum, H. 418 dromedarii, 79 H. excavatum, 62 H. impeltatum, and 301 H. scupense. An integrated YOLOv11-CNN cascade pipeline was designed for tick detection, genus-level filtering against non-Hyalomma arthropods, dorsal/ventral orientation recognition, sex determination, and species-level classification. Performance was assessed using standard classification metrics, YOLO-specific detection metrics, confusion matrices, and LIME-based explainability analysis. The YOLOv11 model reliably detected tick regions, while the taxonomic filtering module discriminated Hyalomma ticks from other arthropod genera, including Argas, Ixodes, Rhipicephalus, and Melophagus. CNN classifiers achieved high performance for sex determination and species recognition, with species-level accuracy ranging from 97% to 100% under controlled laboratory imaging conditions. LIME analysis indicated that model predictions were associated with biologically relevant morphological regions used in classical tick taxonomy. This study provides a first YOLOv11-CNN cascade prototype for automated identification of closely related Hyalomma species and offers a promising decision-support tool for standardized tick identification, veterinary diagnostics, and future One Health surveillance after integreting future studies involving larger and geographically diverse collections will be necessary to further improve model robustness and transferability.
Feline leishmaniosis (FeL) is increasingly recognized in geographical areas where leishmaniosis is endemic. Despite advances in understanding FeL, its diagnosis remains poorly investigated, and the methods currently employed are adapted from canine protocols, overlooking feline-specific biological and pathological features. Here, we evaluated the performance of serological and molecular diagnostic approaches for FeL detection using invasive and non-invasive samples. Based on the estimated minimum sample size, 51 cats were enrolled through convenience sampling. The cats underwent clinical examinations and were tested using parasitological (cytology, histology, and culture), serological Dual Path Platform (DPP®) and Indirect Fluorescent Antibody Test (IFAT), and molecular techniques targeting Leishmania spp. Kinetoplast DNA (kDNA-PCR) and the Internal Transcribed Spacer 1 of ribosomal DNA of Trypanosomatidae (ITS1-PCR). Parasitological methods confirmed Leishmania spp. infection in 7.8% of cats. All parasitologically positive cats exhibited clinical signs consistent with FeL, and infection was confirmed as L. infantum by sequencing. Using parasitological methods combined with clinical signs as the reference standard for confirmed FeL cases, IFAT showed 100% (CI: 40%-100%) sensitivity (Se) and 40% (CI: 26%-57%) specificity (Sp) at a 1:40 cut-off. At a 1:80 cut-off, Se remained at 100% (CI: 40%-100%), and Sp increased to 62% (CI: 46%-76%). DPP® showed higher Sp (95%; CI: 84%-99%). Molecular analysis showed 100% Se and Sp among invasive samples (lymph node, bone marrow, skin, and blood) for both kDNA-PCR and ITS1-PCR, with excellent kappa agreement between assays (κ: 0.898). Among non-invasive samples, conjunctival swabs demonstrated better diagnostic accuracy for both PCR assays, with kDNA-PCR showing improved detection [100% (CI: 40%-100%) Se and 98% (CI: 88%-100%) Sp]. In contrast, oral swabs showed reduced detection capability for both PCR methods, particularly for ITS1-PCR. Our results support the need to standardize serological tools for use in cats. Additionally, the findings support the inclusion of blood and conjunctival swabs as practical alternatives for molecular diagnosis, particularly under field conditions. This study contributes to optimizing diagnostic strategies for FeL in endemic areas and provides practical insights for veterinary diagnosis and epidemiological surveillance.
Neospora caninum is a major cause of abortion and neonatal mortality in cattle worldwide. While vertical transmission is considered the primary route of infection, the precise relationship between maternal parasitemia, placental infection, and congenital transmission remains unclear. This study aimed to investigate the associations between maternal serological and molecular status, placental infection, and neonatal infection in precolostral calves to better understand the mechanisms of vertical transmission. A cross-sectional study was conducted on 53 postparturient clinically healthy Holstein cows and their full-term precolostral, clinically healthy calves from a large dairy herd in Qazvin Province, Iran. Blood samples were collected from both dams and calves immediately after birth, prior to colostrum intake. Placental tissue was sampled from the caruncle-cotyledon junction. N. caninum-specific antibodies were detected by indirect ELISA, and parasite DNA was identified in buffy coat and placental tissues using conventional and nested PCR targeting the NC5 gene. Associations were analyzed using chi-square tests, odds ratios (OR), and Phi coefficients. Overall, 38% (20/53) of dams and 23% (12/53) of precolostral calves were infected. The vertical transmission rate among infected dams was 60%. Maternal infection (by ELISA or PCR) and placental PCR-positivity were significantly associated with calf infection (OR = 21.6-45.4, P-value < 0.001). Significant associations were observed between dam and calf PCR results (φ = 0.63) and between dam and calf ELISA results (φ = 0.52). Kappa analysis showed good agreement between ELISA and PCR in both dams (κ = 0.69) and calves (κ = 0.68). Findings suggest that vertical transmission of N. caninum is substantial and significantly associated with maternal and placental infection status. However, both ELISA and PCR are reliable tools for identifying at-risk animals; their simultaneous use may enhance diagnostic accuracy in control programs.
Sparicotyle chrysophrii is a blood-feeding ectoparasite that poses a significant threat to the farming of gilthead seabream (Sparus aurata), a species of high economic relevance in Mediterranean aquaculture. There is an urgent need for effective, safe, and practical antiparasitic treatments to control these infections. In this study, a phenotypic screening approach was applied to identify compounds active against S. chrysophrii. A panel of reference anthelmintic drugs was selected and used as query molecules in a computational ligand-based virtual screening. The parasiticidal efficacy and potency of the reference drugs, together with compounds selected based on topological indexes, were assessed against adult S. chrysophrii using an in vitro dose-response assay. Among the tested compounds, three salicylanilide derivatives exhibited the highest short-term efficacy, showing the lowest lethal doses 50 (LD50): niclosamide (LD50=0.12 µM), closantel (LD50=2.3 µM), and ME1.62 (LD50=0.09 µM). Niclosamide, one of the most potent compounds identified, demonstrated a parasiticidal activity more than 11,000-times greater than praziquantel (LD50=1.43 mM), a reference chemotherapeutic agent commonly used against fish polyopisthocotylean parasites.
The aim of this study was to assess the possibility of deep learning-based object detection models for the early and comprehensive detection of animal trypanosomosis. We constructed deep learning models for the early detection of Trypanosoma parasites including Trypanosoma congolense from in vitro and in vivo thin blood smears. Our models were based on YOLO (You Look at Once), one of the most common and high-performance object detection models. The method was applied to 14, 380 thin blood smear images with 47, 276 parasites (cells). For the T. congolense, the possibility for the early detection of the parasites was assessed through different concentration levels of cultured parasites (sparse to dense) and time-course blood sampling from infected mice. The in vitro model trained by T. congolense was applied to different species of T. brucei brucei and T. evansi in order to investigate the comprehensiveness of our approach. Our deep learning models successfully identified Trypanosoma parasites even for the settings of early detection. Our models also showed high precision (>0.90) for the dense and late predictions, not only for the same species and same sample source (in vitro / in vivo) of trypanosomes but also for the different species and different sample source (comprehensive prediction from in vitro to in vivo). The results showed that our methods are applicable for the purpose of early detection, not only for a specific Trypanosoma parasite spp. and the same sample source, but also for other spp. and sample source.
Tropical theileriosis, caused by the tick-transmitted apicomplexan parasite Theileria annulata, remains a major constraint on cattle production across North Africa, the Mediterranean basin, the Middle East and South Asia. Current control depends on acaricides, the theilericidal drug buparvaquone and live attenuated schizont vaccines, but acaricide resistance, buparvaquone-resistance mutations and the logistical demands of vaccination are eroding the sustainability of these tools. Host genetics offers a complementary and durable alternative. Indigenous Bos indicus breeds are consistently more resistant to ticks and tolerate T. annulata infection better than exotic Bos taurus cattle, and this advantage has a measurable heritable component. Unlike previous reviews, which treat tick resistance, T. annulata immunobiology and livestock genomic selection as separate subjects, we integrate all three and assess host genetics specifically against the failure modes of current control. We review the tick, parasite and host interface, the evidence for natural resistance, and the genetic and immunological mechanisms involved, including signal-regulatory protein, bovine major histocompatibility complex class II and inflammatory pathway genes. We then assess whether genomic selection, multi-omics, machine learning and gene editing can translate these mechanisms into resistant cattle, and we weigh the biological, economic and infrastructural barriers to implementation. The evidence indicates that host genetics will not replace existing control but could reduce reliance on acaricides and chemotherapy. That contribution remains prospective rather than demonstrated: no resistance marker for T. annulata has yet been validated, prediction accuracies are moderate and transfer poorly between breeds, and no endemic production system has implemented selection for resistance.
Avian coccidiosis, caused by Eimeria spp., remains a major parasitic disease of poultry and imposes significant economic burdens on the global poultry industry. This review systematically synthesizes key advances over the past decade concerning host-Eimeria interactions, molecular regulatory mechanisms, and novel control strategies, while contextualizing these findings with earlier seminal discoveries. In recent years, novel diagnostic tools based on molecular detection and antigen capture have emerged, offering improved sensitivity and interspecies specificity over conventional methods. These techniques complement traditional approaches relying on oocyst morphology and histopathology, and provide critical support for accurate assessment of field infection status, species and genotype discrimination, monitoring of drug-sensitivity shifts, and elucidation of transmission dynamics. Epidemiological investigations have further revealed the impacts of rearing management, environmental temperature and humidity, host genetic background, and gut microbiota composition on infection kinetics, underscoring the necessity of integrating biosecurity and precision management into regionally tailored control programs. Utilizing chicken embryo and chick infection models, in conjunction with CRISPR/Cas9 gene editing, single-cell transcriptomics, and high-resolution proteomics, researchers have gained deeper insights into key regulatory genes governing invasion, asexual multiplication, and gametogenesis, as well as invasion-related effector molecules and resistance-associated markers, thereby laying a foundation for the identification of novel intervention targets. In immunology, growing knowledge of the intestinal epithelial barrier response, Th1/Th17 polarization, regulatory T-cell function, and immune evasion strategies (e.g., antigenic variation and downregulation of host antigen presentation) provides a theoretical basis for the rational optimization of subunit vaccines and live oocyst vaccines. On the therapeutic front, novel combination regimens of conventional anticoccidials and plant-derived bioactive compounds have shown efficacy in reducing oocyst shedding and alleviating intestinal lesions, while nanoparticle-based targeted delivery systems and adjuvant combination strategies are being explored to enhance drug bioavailability or vaccine-induced protective immunity. Nevertheless, the effective integration of ever-expanding omics data, immune-protective mechanisms, and field-applicable control measures, while concurrently addressing drug residues and resistance management, remains a central challenge for achieving sustainable coccidiosis control.
Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by infection with Echinococcus granulosus sensu lato, with the liver being the primary affected organ. This study aimed to investigate the metabolic profile alterations and differential regulation of the PPARγ signaling axis in proximal and distal hepatic tissues following E. granulosus(s.l.) infection in sheep, thereby elucidating the molecular mechanisms underlying local lipid metabolic reprogramming. Naturally E. granulosus(s.l.) infected sheep livers were used as subjects, with cyst-adjacent Close liver tissue (CLT), Distal liver tissue (DLT), and uninfected liver tissue (NC) collected as controls. Untargeted metabolomics combined with KEGG enrichment analysis was employed to compare metabolic profiles among the three groups; GSEA enrichment analysis was performed to assess changes in lipid metabolism-related pathways; qPCR and Western blot were used to detect mRNA and protein expression levels of PPARγ, SREBP-1c, ACC1, and CD36; HE staining was conducted to observe the degree of inflammatory infiltration. Metabolomic analysis revealed that, compared with the NC group, the CLT group exhibited suppressed TCA cycle activity, amino acid metabolism disorders, and purine metabolism abnormalities, whereas these disturbances were markedly attenuated in the DLT group. KEGG and GSEA enrichment analyses further demonstrated that the PPAR signaling pathway, steroid hormone biosynthesis, and arachidonic acid metabolism were significantly downregulated in the CLT group, with alterations in glycerophospholipid metabolism also observed. At the molecular level, mRNA and protein expression levels of PPARγ, SREBP-1c, ACC1, and CD36 in the CLT group were significantly lower than those in the NC group, while these parameters showed substantial recovery in the DLT group. HE staining indicated that the degree of inflammatory infiltration in the CLT group was significantly higher than that in the DLT group. E. granulosus(s.l.) infection can suppress the PPARγ signaling axis in proximal hepatic tissues of sheep, leading to lipid metabolic reprogramming and exacerbated inflammation, whereas distal tissues can maintain metabolic homeostasis through compensatory mechanisms. Purine metabolism disorders may attenuate PPARγ and downstream gene expression by regulating AMPK signaling, providing novel therapeutic targets for metabolic intervention in CE-associated liver injury.
Detection of Dirofilaria immitis antigens is a cornerstone for diagnosis of heartworm disease (HWD). However, no antigen detected (NAD) results may occur secondary to various biological phenomena, including antigen-blocking (AB). Serum heat treatment has been proposed as a strategy to dissociate circulating immune complex (CIC) and reverse AB. This study aimed to: (i) determine the frequency of NAD results; (ii) evaluate the efficacy of heat treatment in reversing NAD results; (iii) investigate potential associations between AB, immune response and seropositivity to other vector-borne pathogens (VBPs). Blood and serum samples were collected from 101 D. immitis-infected dogs from Sardinia (n = 73) and Sicily (n = 28), as well as from 7 healthy dogs serving as controls. Dirofilaria immitis microfilariae and antigens were detected using Knott's test and IDEXX SNAP Leish 4Dx®, respectively. Seropositivity with Anaplasma spp., Ehrlichia spp. and Leishmania infantum, was evaluated simultaneously. Dirofilaria immitis antigen-negative serum samples underwent heat treatment. Serum concentrations of interferon gamma (IFN-γ), interleukin 12 (IL-12), and interleukin 4 (IL-4) were quantified via enzyme-linked immunosorbent assays (ELISA) across three groups: antigen-blocked dogs (AB, n = 8), SNAP-positive/Knott-positive dogs (DC, n = 20), and healthy non-infected dogs (C, n = 7). Initial antigen negativity was observed in 11 out of 101 samples (10.9%), with heat treatment successfully reversing NAD in 8 of the 11 cases (72.7%) yielding an overall confirmed AB frequency of 7.9% (8/101). Interleukin analysis revealed significantly higher IL-4 levels in the AB group [median 53.71 pg/mL (Q1-Q3: 35.94-71.00); P < 0.001] and significantly higher IL-12 levels in the DC group (49.85 ± 14.56 pg/mL; P = 0.001). Overall VBPs seropositivity was significantly higher in the AB group (4/8, 50.0%) than in the DC group (2/20, 10.0%; P = 0.0384). Specifically, L. infantum seropositivity was detected in 37.5% (3/8) of AB dogs compared to 5.0% (1/20) of DC dogs. Antigen blocking emerged as a relevant cause of NAD results in canine HWD, occurring in approximately 8% of infected dogs in this study. Heat treatment successfully restored antigen detection in most NAD samples. The association between antigen blocking and increased IL-4 concentrations, potentially amplified by co-positivity with other VBPs, suggests that a Th2-biased immune response may contribute to CIC formation and subsequent antigen masking.
Coccidiosis is a disease of major importance in poultry husbandry, and the current control measures such as chemoprophylaxis and live vaccines are not sustainable. Therefore, there is a need for sustainable alternative approaches. It has been suggested that biochar can bind to the Eimeria parasites that cause coccidiosis and inhibit their replication in the gut. The aim of this study was to evaluate the impact of dietary biochar on growth rate, infection outcome, caecal microbiota, and specific immune response of broilers experimentally infected with E. tenella. A total of 64 day-old chicks were randomly assigned to one of two dietary treatments; control or biochar. Chickens were fed the biochar diet (2% inclusion rate) from arrival and until the end of trial when they were 30 days old. Eimeria. tenella inoculation was performed when chickens were 20 days old. We found that dietary biochar did not inhibit the E. tenella infection of the caecum demonstrated by similar oocyst shedding, lesion scores and growth rates in the control and biochar groups. Furthermore, biochar did not significantly affect the different blood leukocyte populations monitored or induction of E. tenella specific immune responses. However, biochar caused decreases in the abundance of potentially beneficial bacterial families such as Lactobacillaceae and Bifidobacteriaceae, and an increase of the fungal family Aspergillaceae in the caecum. Therefore, supplementing feed with biochar may be an unsuitable or even a contraindicated approach to prevent E. tenella infection in chickens.
The development of anthelmintic resistance due to long-term and irregular use of drugs such as ivermectin (IVM) poses a major challenge to the prevention and control of haemonchosis caused by Haemonchus contortus. However, the mechanism of IVM resistance in H. contortus remains incompletely understood. Reactive oxygen species (ROS) generated under oxidative stress conditions serve as key regulators of autophagy. Reactive oxygen species induce autophagy, and autophagy mitigates oxidative stress-mediated damage, thereby enhancing cell survival. Although autophagy is known to contribute to H. contortus resistance, the involvement of ROS and autophagy in H. contortus resistance remains unclear. This study explores the regulatory role of ROS-mediated autophagy in H. contortus resistance to IVM, using sensitive and resistant strains as experimental subjects, with resistance assessed via larval migration inhibition tests and RT-qPCR, autophagy levels detected by RT-qPCR and transmission electron microscopy, and ROS levels determined by the DCFH-DA fluorescence probe assay. Results showed that baseline ROS levels were higher in the resistant strain than in the sensitive strain, and IVM treatment increased ROS levels in the sensitive strain (P < 0.05). After N-acetyl-L-cysteine (NAC)-mediated inhibition of ROS, autophagy levels and IVM resistance in the resistant strain were substantially reduced. Lipopolysaccharide (LPS)-induced upregulation of ROS led to substantial increases in autophagy levels and IVM resistance in the sensitive strain. The study demonstrates that ROS may enhance H. contortus resistance to IVM by inducing autophagy. This research shows a potential relationship between ROS, autophagy, and IVM resistance, providing insights into the mechanisms underlying resistance in H. contortus.
Experimental infection of a host with nematodes requires embryonated eggs. Embryonable eggs can be isolated either directly from the uteri of adult worms or from host faeces. This study investigated whether mature female Ascaridia galli worms recovered from the intestines of naturally infected laying hens can produce and release substantial numbers of eggs during a short off-host incubation, and whether such an incubation reduces uterine egg reserves. Seventy mature female A. galli worms were allocated to 14 replicate batches of five worms. Seven replicates were processed immediately for uterine egg recovery, while the remaining seven replicates were incubated in phosphate-buffered saline at 40 °C for 48 h. Nematode eggs released into the medium were quantified after 24 and 48 h, after which uterine eggs were recovered from the incubated worms. Egg counts from uteri of worms processed immediately at day 0 had an average of 32,420 (SD = 15,369) eggs per female with no significant difference (P = 0.544) from the uterine eggs of those processed after 48 h of incubation which had 29, 942 (SD =10,010) eggs. The incubated worms released 7548 (SD = 4438) eggs per female during day 1 and 7739 (SD = 3369) eggs per female during day 2, with no significant difference between the two days (P = 0.921). The daily egg release represented 28.3-36.4% of the uterine egg reserves, suggesting that mature female A. galli can release approximately one-third of their uterine eggs within 24 h as fully mature eggs. We conlcude that mature A. galli females can maintain short-term egg release outside the host without depletion of uterine egg reserves. The short-term off-host incubation offers a practical means of increasing egg recovery from a limited number of adult worms, and may thus contribute to reducing the number of chickens and worms required for the preparation of A. galli infection material.
