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.
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.
Anaplasma marginale, Babesia bovis, and Babesia bigemina are hemoparasites that can co-infect ruminant livestock and cause symptoms that result in substantial economic loss and even death. These parasites are carried by ticks, which bite the host and transfer the hemoparasite into the blood. The standard approach to identify hemoparasites is Giemsa-stained thin blood smears observed under a microscope, but it is time-consuming and can be difficult to identify co-infections. Thus, we developed a novel triplex PCR to simultaneously detect B. bovis and B. bigemina (based on 18S ribosomal DNA [18S rDNA]) and A. marginale (based on the major surface protein 4 [MSP4] gene). The specific primers produced an amplicon of 168 base pairs (bp) for B. bigemina, 267 bp for B. bovis, and 478 bp A. marginale. The triplex PCR required a minimum of 0.1 pg of DNA to identify B. bovis, B. bigemina, or mixed infection, and 1 pg of DNA to identify A. marginale. There was no cross-amplification with Theileria spp. and Trypanosoma evansi DNAs. The triplex PCR could successfully amplify the three DNA targets simultaneously in cattle blood samples (n = 114) from Thailand. The triplex PCR detected more positive samples (27/114; 23.68%) than microscopic examination (5/114; 4.39%) for all three hemoparasites. Our study offers an alternative, specific, sensitive, and accurate identification and detection tool for co-infection of B. bovis, B. bigemina, and A. marginale in bovine blood. The developed triplex PCR assay could be useful for routine diagnostics in veterinary laboratories and for epidemiological monitoring of hemoparasites.
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.
Ticks are haematophagous ectoparasites of major veterinary and public health significance. Traditionally, tick rearing has relied on live animal hosts for studying tick biology, pathogen transmission and the discovery of drugs and vaccines but increasing concerns regarding animal welfare have driven the development and refinement of artificial tick feeding systems (ATFS). Here, we demonstrate the first successful consecutive artificial feeding of larvae, nymphs and adults of Rhipicephalus australis using an optimised silicone membrane system. Larval attachment and engorgement rates were 61.8 ± 20.4% (range 30.3-95.1%) and 40.1 ± 12.4% (24.0-61.0%), respectively. Nymphs exhibited the highest feeding success, with attachment and engorgement rates of 88.9 ± 8.5% (69.2-98.2%) and 91.8 ± 9.5% (66.7-100.0%), respectively, demonstrating the viability and fitness of nymphs that had moulted from artificially fed larvae. Adult ticks successfully attached (83.7 ± 5.9%; 76.9-87.5%) and engorged (89.1 ± 4.2%; 84.6-92.8%) and yielded viable engorged females. However, oviposition was not observed, and antimicrobial supplementation was required to control microbial contamination. Therefore, this system represents an important initial step towards controlled feeding across all stages of R. australis. Further optimisation, particularly to improve reproductive performance and reduce reliance on antimicrobials, will be required before its broader application to tick-pathogen interaction and vector competence studies.
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.
Sarcocystis aucheniae is a coccidian parasite of South American camelids (SAC) that causes substantial economic losses through carcass condemnation due to the presence of macroscopic muscular cysts. Although horizontal transmission through canid definitive hosts is well established, the remarkably high prevalence of infection observed in alpacas and llamas suggests that additional transmission routes may contribute to parasite maintenance. The aim of this study was to investigate the occurrence of S. aucheniae DNA in placenta and umbilical cord tissues. Placenta and umbilical cord samples were collected from pregnant alpacas and llamas in the final third of gestation at a slaughterhouse in southern Peru, as well as from alpacas sampled at parturition in a breeding establishment. DNA was extracted and analyzed using a previously described duplex semi-nested PCR targeting the S. aucheniae 18S rRNA gene. Selected positive amplicons were subjected to Sanger sequencing. Parasite DNA was detected in at least one tissue (placenta and/or umbilical cord) in 33 of 42 animals (78.6%). Parasite detection in placenta and umbilical cord was not statistically significantly different in paired samples, and animals positive in umbilical cord samples had increased odds of testing positive in placenta. Agreement between tissues was moderate, suggesting an uneven distribution of parasites within maternal-fetal compartments. No association was observed between parasite detection and age category. Sequencing confirmed the identity of the amplified fragments as S. aucheniae. Although detection of parasite DNA in placental and umbilical cord tissues does not by itself demonstrate fetal infection, the high frequency of positive animals and the significant association between both tissues provide the first molecular findings consistent with possible vertical transmission in S. aucheniae. These findings broaden current understanding of the parasite life cycle and suggest that vertical transmission may contribute to parasite persistence in SAC populations, with important implications for disease control.
Ctenocephalides felis felis is the most prevalent tick species infesting domestic animals and represents an important target for sustainable ectoparasite control. This in vitro study investigated the insecticidal and repellent activity of essential oils from Cymbopogon nardus, Cymbopogon winterianus, Cymbopogon flexuosus, and Cymbopogon martinii, as well as the monoterpene geraniol, against all developmental stages of cat fleas. The chemical composition of the essential oils was determined by gas chromatography with flame ionization detection. Insecticidal activity was evaluated using a contact bioassay involving filter paper impregnated with different concentrations of the essential oils and geraniol. Lethal concentrations were estimated using Probit analysis. Residual efficacy was assessed by exposing adult fleas to treated filter paper and recording mortality at 24-hour intervals, and repellency was evaluated by comparing the distribution of adult fleas between treated and control filter paper strips. All treatments showed concentration-dependent activity. Geraniol presented the lowest LC₅₀ values for eggs (14.7 μg.cm⁻²), pupae (47.1 μg.cm⁻²), adults (84.6 μg.cm⁻²), and biological cycle inhibition (3.6 μg.cm⁻²), whereas C. martinii essential oil was the most potent against larvae (LC₅₀ 19.4 μg.cm⁻²). With regard to residual adulticidal activity, C. martinii essential oil and geraniol-maintained activity for approximately 25-26 days, whereas C. nardus, C. winterianus and C. flexuosus lost activity after approximately 19-20 days. Repellency at 800 μg.cm⁻² was pronounced for all essential oils during the first hours, with C. martinii essential oil maintaining ≥ 90% repellency from 1 to 48 h, and geraniol from 1 to 12 h. These findings indicate that Cymbopogon essential oils, particularly C. martinii, and geraniol have significant in vitro insecticidal and repellent activity against C. felis felis.
Outbreaks of trypanosomiasis in cage-cultured large yellow croaker in China has caused significant economic losses, however, to date, there are no effective drugs available for the prevention and treatment of this disease. Metronidazole is a nitroimidazole derivative with a wide range of biological activity ranging from antiparasitic to antibacterial, and is especially useful for the treatment of anaerobic protozoan infections. Nevertheless, studies on its efficacy against protozoan parasites in aquatic animals are lacking. In this study, an efficacy trial of metronidazole against trypanosomes isolated from large yellow croaker was conducted, based on the established infection model using largemouth bass, with an intraperitoneal injection dose of 10⁵ cells/mL (100 μL per fish) and maintained at 25°C. The in vivo anti-parasitic efficacy results showed that 300 mg/kg metronidazole significantly reduced the trypanosomes load, resulting in complete parasite clearance in most fish (88.9%) and prevented mortality of fish (95.56%). Acute toxicity test results indicated that 1500 mg/kg metronidazole remained safe for largemouth bass. Meanwhile, histopathological analysis revealed that in the 300 mg/kg metronidazole-treated group, the number of red blood cells increased and their morphology normalized in the liver, spleen, and head kidney. Besides, enzyme activity assays proved the differential expression of alanine aminotransferase (ALT), acid phosphatase (ACP), and total superoxide dismutase (T-SOD) among the positive control (PC), negative control (NC), and metronidazole-treated groups. qPCR results showed that metronidazole can increase the expression level of TNF-α, IFN-γ, and IL-1β, and IL-10. In conclusion, this study demonstrated the promising efficacy of metronidazole against fish trypanosomiasis, laying a solid theoretical and practical basis for controlling aquatic animal parasitic diseases.
Fasciolosis is a zoonotic parasitic disease caused by liver flukes Fasciola hepatica and Fasciola gigantica, which is distributed worldwide, causes significant losses to livestock farming. The development of a rapid detection method will aid in disease prevention and control. Although there are many studies on rapid diagnostics, most are based on natural antigens. A colloidal gold detection method based on recombinant antigens for diagnosing F. gigantica infection is lacking. Given the potential of mixed recombinant antigens for enhanced diagnostic efficacy, a colloidal gold method based on recombinant mixed antigens is established in this study. An indirect ELISA based on mixed recombinant FgSAP-2 (rFgSAP-2) and FgCatL1 (rFgCatL1) was developed and assessed. Then a colloidal gold immunochromatographic test strip against F. gigantica infection (FgICS) based on mixed rFgSAP-2 and rFgCatL1 was further developed, and the sensitivity, cross-reactivity and accuracy of the established FgICS was assessed by serum samples. When testing 300 field serum samples, the mixed-antigen ELISA yielded a positive rate of 75.00%, which was higher than ELISAs coated with antigens rFgSAP-2 (60.67%), rFgCatL1 (63.00%) and F. gigantica excretory and secretory protein (FgESP) (65.33%). Subsequently, a colloidal gold test strip labeled with Staphylococcus aureus protein A (SPA) based on mixed antigens was also developed, remained positive at a serum dilution of 1:1280, with no cross-reactivity. Testing of 100 serum samples yielded an overall accuracy of 89% for FgICS when referenced against the 4-ELISA consensus standard. An ELISA based on mixed antigens FgSAP-2 and FgCatL1 showed better sensitivity and stability. The newly developed mixed antigens colloidal gold strip also retained high sensitivity and accuracy, which can be used for development of point‑of‑care test kits and provides a theoretical basis for the diagnosis and control of F. gigantica infection.
Babesia (B.) naoakii, previously referred to as Babesia sp. Mymensingh, is a recently characterized tick-borne haemoprotozoan parasite of cattle. In Indonesia, we first reported its presence in 2022 from clinically affected cattle in Central Java. To investigate the wider epidemiology of this neglected ruminant-associated Babesia species, we surveyed apparently healthy cattle (Bos indicus) and water buffaloes (Bubalus bubalis) across three districts of Java, Indonesia. A PCR assay targeting the B. naoakii-specific apical membrane antigen 1 (ama1) gene detected the parasite occurrence in 34.39% of assessed cattle (87/253; 95% CI: 28.80-40.44%) and 30.77% of water buffaloes (12/39; 95% CI: 18.47-46.52%). These results represent the first record of B. naoakii infection in water buffaloes in the country and confirm that the parasite circulates in subclinically infected bovine hosts. To characterise this apicomplexan parasite further at the molecular level, we assembled in full length the three mitochondrial protein-coding genes (PCGs): cytochrome c oxidase subunits 1 (cox1) and 3 (cox3), as well as cytochrome b (cytb). These genes were reconstructed by next-generation sequencing of blood DNA collected during the acute haemolytic-phase of B. naoakii infection, from calves that subsequently succumbed to the disease in the endemic area. Phylogenetic analyses of the concatenated amino-acid sequences of cox1, cox3, and cytb placed the Indonesian isolates within a well-supported monophyletic clade, distinct from all previously characterised ruminant-associated Babesia species and sister to the Babesia bigemina/Babesia ovata lineage. This placement confirmed species identity and reinforced the genetic distinctiveness of B. naoakii in Indonesia. Notably, although B. naoakii circulates in peripheral blood and mirrors the diagnostic behaviour of the mild pathogen B. bigemina, its clinical impact more closely resembles that of the severe pathogenic B. bovis, particularly in young animals. This diagnostic-clinical discordance highlights the need for B. naoakii-specific molecular surveillance and species-level differentiation in regions of co-endemicity. Given the high prevalence in subclinically B. naoakii-infected adults, the documented severity of babesiosis in calves, and the potential for substantial economic losses, broader epidemiological investigations and species-specific control measures for B. naoakii are urgently performed. The same holds true for future epizootiological investigations of underdiagnosed B. naoakii-infections possibly circulating in Indonesian endemic ruminant bovids such as the banteng (Bos javanicus), the lowland anoa (Bubalus depressicornis) and the tamaraw (Bubalus mindorensis).
This study describes, for the first time, the molecular and phylogenetic detection of Babesia bovis in sheep, with concurrent detection in cattle and R. microplus ticks during a severe tick infestation outbreak in northeastern Brazil. The affected herd, located in São Pedro, Rio Grande do Norte State, comprised sheep and cattle raised under a semi-intensive system. Blood, fecal, and tick samples were collected from 20 sheep and three cattle. Blood samples were subjected to complete blood count analysis and, together with tick samples, to DNA extraction and molecular detection of Anaplasma marginale, B. bovis, and Babesia bigemina. Amplified fragments were sequenced and analyzed phylogenetically. The sheep exhibited heavy tick infestations, alopecia, and skin lesions. Hematological analysis revealed anemia in 8/20 of the sheep, leukocytosis in 5/20, and thrombocytopenia in one animal. Coproparasitological examination revealed a high burden of nematode eggs of the order Strongylida in 12/20 of the evaluated sheep. Molecular analysis detected B. bovis in one sheep, one bovine, and ticks collected from sheep. Only cattle were positive for B. bigemina, whereas A. marginale was not detected in any sample. The sequence obtained from the sheep shared > 97% identitity with B. bovis isolates available in GenBank and clustered with the bovine and tick isolates from the same farm, indicating a close genetic relationship and supporting the hypothesis of local circulation of the parasite. The observed anemia was likely multifactorial, associated with tick infestation, gastrointestinal parasitism, and possibly B. bovis infection. This study provides molecular and phylogenetic evidence of the occurrence of B. bovis in sheep, cattle, and R. microplus ticks during a heavy tick infestation outbreak, suggesting the circulation of a common parasite lineage and a possible epidemiological association under field conditions.
