Ticks are important vectors of pathogens with substantial medical and veterinary relevance, a threat that may be further increased with the spread of invasive tick species. Reliable tick identification is relevant for accurate diagnostic elucidation of transmission pathways, and the development of targeted surveillance strategies and risk assessments. A standardized, rapid, and cost-effective identification workflow that can be implemented across laboratories is needed. Previous studies have shown that MALDI-TOF MS is a promising, objective, and economical approach for tick identification. However, the methodology remains insufficiently standardized and is not yet routinely applied for vector identification. The aim of this study was therefore to optimize MALDI-TOF MS-based tick identification for routine use in diagnostic laboratories. A simplified and standardized protocol for protein extraction from whole tick legs via brief incubation in 70% formic acid is presented. This protocol provides a robust basis for the development of a curated MALDI Biotyper® reference library, enabling reliable, expert-independent tick identification and supporting epidemiological surveillance of vectors and vector-borne pathogens.

Ixodes ricinus is the most widespread tick species in Europe, parasitizing a broad range of vertebrate hosts, including reptiles. However, the role of lizards in the ecology of tick-borne pathogens remains poorly understood. This study investigated tick infestation levels and the occurrence of tick-borne pathogens in I. ricinus feeding on European common lizards (Zootoca vivipara louislantzi) in the central Pyrenees, Spain. A total of 137 lizards were captured in summer and spring of 2024 at two sites (~ 1700 m.a.s.l.). Tick infestation prevalence was 98.5% and a mean abundance of 5.8 ± 7.0 ticks. Infestation was significantly higher in males, adults, and during spring. Molecular screening of 525 ticks (188 pools) using real-time and conventional PCR protocols detected Rickettsia spp. DNA in 15.4% pools, with an overall minimum infection rate (MIR) of 5.52%, with stage-specific MIRs of 4.79% in larvae and 7.81% in nymphs. Sequencing of 14 amplicons identified Rickettsia helvetica (n = 11) and Rickettsia monacensis (n = 3). Babesia DNA was detected at low prevalence (MIR: 0.38%) and sequences clustered within the Babesia divergens-Babesia capreoli complex. No Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA was detected. Our findings show that lizards can sustain high levels of I. ricinus infestation, constituting a reliable bloodmeal source for ticks infected with Rickettsia and Babesia in the mountain ecosystems of the Pyrenees. Although the lizard's role in pathogen transmission remains to be clarified, its consistent exposure to immature ticks highlights its potential value as a sentinel species for monitoring tick-borne pathogen circulation in high-altitude environments.

Theileria annulata, an apicomplexan parasite, causes lymphoproliferative disease in cattle and threatens the bovine industry in tropical and subtropical regions. To clarify life-cycle mechanisms and parasite-host interactions, we conducted high-resolution Orbitrap proteomic and phosphoproteomic analyses of the schizont and piroplasm stages. In total, we confidently identified and quantified 2,093 proteins; among these, 459 proteins were observed in a phosphorylated state, comprising 5,069 distinct phosphorylation sites. Functional enrichment analysis of differentially abundant proteins indicated stage-associated remodeling of genetic information processing, carbohydrate, lipid, and amino-acid metabolism, and core cellular processes. Stage-associated proteins were linked to host-parasite interaction, mitochondrial function, and metabolic adaptation. Schizonts showed broader proteomic and phosphorylation profiles consistent with higher biosynthetic and energy demands. In contrast, piroplasms exhibited a more restricted proteomic profile associated with erythrocyte adaptation and invasion-related functions. Together, these stage-resolved datasets provide a reference for proteome and phosphoproteome remodeling across development and prioritize candidate stage-associated proteins and phosphosites for future functional validation.

Fish serve as hosts to a wide range of parasites, which can affect them in many ways, including their survival, growth, and foraging behavior. The highly threatened unionid mussels have an ectoparasitic life stage on the gills, skin, or fins of fish. Here, we compared prey handling time and growth between groups of brown trout (Salmo trutta) that were infested and non-infested with glochidia larvae of the freshwater pearl mussel (Margaritifera margaritifera). In a prey handling test, individual trout were presented with one large prey (maggot) and one small prey (Gammarus pulex) daily over eight days. Compared to non-infested trout, prey handling time was longer for infested trout feeding on larger but not on smaller prey. In a growth test, where trout were fed daily with minced calf liver over nine weeks, the growth rate was lower for infested than for non-infested trout. Moreover, the growth rate was higher for infested trout from which glochidia were expelled compared to trout from which glochidia were not expelled. We suggest that glochidia infestation can increase prey handling time of fish, ultimately reducing fish growth, but that slow prey handling may be transitory for fish losing glochidia, and result in compensatory growth of the fish. Slow prey handling, in combination with a negative effect of glochidia infestation on food competition between infested and non-infested trout, may be reasons behind the lower growth of infested fish.

The most prevalent approach during treatment of cystic echinococcosis (CE) relies on surgical cyst removal and long-term chemotherapy with benzimidazoles. However, cyst rupture may lead to anaphylaxis or secondary cysts and adverse effects. This study is aimed at synthesizing and evaluating the potential of clove oil-loaded solid lipid nanoparticles (ClO-SLNs) against Echinococcus granulosus protoscoleces. Clove bud (Syzygium aromaticum) oil was purchased and ClO-SLNs were synthesized. Physicochemical characterization of the nanoparticles was performed using transmission electron microscopy (TEM) and their zeta potential was measured. In addition, encapsulation efficiency (EE%) and drug loading (DL%) were performed. The cytotoxicity of different concentrations of ClO-SLNs was evaluated on HepG2 cell line. Protoscolicidal effects of different concentrations of ClO-SLNs were investigated as well. One-way ANOVA followed by post hoc tests where appropriate was employed to analyze data. Nanoparticles with round smooth margins were characterized by TEM. The size of particles was about 122.3 ± 30.0 nm with a zeta potential of -0.9 mV. The EE% and DL were 59.68% and 16.08%, respectively. The cell viability remained high (> 80%) across two concentrations, 100 μg/mL and 1 mg/mL, of ClO-SLNs. Raw clove oil extract exhibited no cytotoxicity at 50 mg/mL and the cell viability of albendazole was more than 60% at 10 mg/mL. The mortality rates of protoscoleces exposed to ClO-SLNs were concentration-dependent in all time points with p values: 0.013, 0.0074, 0.0064, and 0.0042 for 15, 30, 60, and 120 min. The ClO-SLNs exhibit good biocompatibility across all tested concentrations with highly potent and rapid protoscolicidal activity against E. granulosus protoscoleces. These findings suggest that ClO-SLNs may represent a potential protoscolicidal candidate for future intraoperative applications. However, further in vivo studies are required before considering clinical evaluation.

Malaria remains a major public health problem in Ethiopia, with heterogeneous transmission across ecological and demographic settings. This study assessed variation and demographic determinants of malaria infection in selected villages of Bibugn woreda, Amhara, Ethiopia. A community-based cross-sectional survey was conducted from October to December 2016 and March to April 2017 in Ameba Amarsa, Debersina, and Moseba Shemiabo villages, which were purposively selected from the woreda. A total of 422 residents were randomly selected from the purposively selected villages. Thick and thin blood smears were examined microscopically to detect Plasmodium species. Data were entered and analyzed by using SPSS software. Associations between malaria infection and predictor variables were analyzed using chi-square tests at a 95% confidence level. Overall, 46 participants were positive, yielding a malaria prevalence of 10.9% (95% CI: 8.0%-14.3%). Infection was higher among males (67.39%) than females (32.61%), but not statistically significant (χ [2] = 1.753, df = 1, p = 0.185). The occurrence of Plasmodium species was not significant in residence (χ [2] = 3.609, df = 1, p = 0.058), but there is significant variation in age (χ [2] = 9.909, df = 2, p = 0.007). Plasmodium vivax was the predominant species, accounting for 60.87% (28/46) of infections, whereas P. falciparum comprised 39.13% (18/46). Debersina village exhibited the highest proportion of infections (69.56%), followed by Moseba Shemiabo (17.39%) and Ameba Amarsa (13.04%). Malaria transmission persists at a moderate level in Bibugn Woreda, with P. vivax emerging as the dominant parasite contrasting the national pattern of P. falciparum predominance. Strengthening surveillance, improving vector control coverage, and incorporating P. vivax specific strategies are critical for accelerating malaria elimination efforts in the district.

Varroa destructor (V. destructor) threatens honeybee colonies and negatively affects drone weight and survival. Therefore, this mite species poses a risk to the reproductive potential of drones, and subsequently colony health and performance. However, studies on the impact of V. destructor infestation on sperm characteristics are limited, and results appear to be inconclusive. This study aimed to investigate the effect of V. destructor infestation on multiple sperm quality characteristics that are regarded as important for successful fertilization. In this cross-sectional study, conducted in the Western Cape, South Africa, during the summer of 2021, 53 sexually mature Apis mellifera capensis (A.m. capensis) were harvested. Analysis of semen included functional (total motility, progressive motility and swimming speeds; kinematic parameters) and structural sperm parameters (sperm head, tail, total length) using a computer-aided sperm analysis system. Drone weight and demographic information of colonies were also assessed. Three infestation groups were compared, i.e., < 1% (n = 4 colonies) (median: 0.33%; range: 0.00-0.40%), 1%-2% (n = 2 colonies) (median: 1.07%; range: 1.00%-1.45%) and 10% (n = 2 colonies) (10.0% ± 0.00%). In this study, V. destructor infestation had minor, non-significant effects on drone weight (11% lower in highly infested drones; p = 0.123) and sperm concentration (p = 0.064). Sperm motility was, however, significantly affected by infestation levels, with the least infected drones producing sperm with significantly higher total motility (99.1% vs. 83.9% and 68.2%; p = 0.002), progressiveness, swimming speed and kinematics (VCL, VSL, VAP; p < 0.001). Sperm vitality was not affected by V. destructor infestation (p = 0.559). Infestation altered sperm structure with drones from highly infected colonies producing significantly longer sperm (tail: 227 ± 1.22 μm vs. 224 ± 2.31 μm; total length: 236 ± 1.23 μm vs. 233 ± 2.16 μm; p < 0.001). Furthermore, significant relationships were observed between V. destructor infestation and sperm functionality as well as sperm structure. Colony demographics was not negatively affected by V. destructor infestation level, but in contrast, significant relationships were found between colony demographics and sperm motility and kinematic parameters as well as sperm structure. Findings of this study suggest that V. destructor infestation may hinder drone reproductive potential through subtle alterations in sperm quality, particularly sperm motility and kinematics. This study further highlights the importance of sperm quality parameters as markers of the impact of V. destructor infestation on honeybee drone and colony reproduction and survival.

The Asian longhorned tick, Haemaphysalis longicornis, is a well-known vector of several zoonoses including SFTS and Japanese spotted fever. This species consists of two different reproductive forms, bisexual and parthenogenetic, which are suggested to have differential roles in disease transmission. In Japan, the distributions of these forms have been suspected from sex bias in each population. Meanwhile, recent insights about the origin of the parthenogenetic form suggest the possibility of individual-level diagnosis using mitochondrial phylogeny. To reassess the current distribution of those reproductive forms and potential changes over time in Japan, 1750 H. longicornis collected across 23 prefectures of Japan were investigated. The COI barcoding region was used to distinguish the mitochondrial haplogroup of each individual. Additionally, we developed real-time PCR probes to distinguish the two mitochondrial haplogroups representing each reproductive form. As previous studies have suggested, the parthenogenetic mitochondrial haplogroup was present throughout the country, while the bisexual mitochondrial haplogroup was found only in central and western Japan. Interestingly, populations consisting of either one or both haplogroups were found in neighboring areas, highlighting the need for more detailed and fine-scaled investigations in order to fully reveal the distribution of the reproductive groups. The newly developed real-time PCR method successfully differentiated all tested haplotypes. Furthermore, alignment of 173 COI haplotypes revealed that the diagnostic nucleotide selected for the method was consistent across all haplotypes, including those outside of Japan. This suggests that this method can accurately distinguish between the two haplogroups of H. longicornis in both Japan and other countries. This one-step method will serve as a rapid tool to distinguish bisexual and parthenogenetic H. longicornis, making further investigations, particularly into their role in disease transmission and shifts in distributions, possible.

Fresh vegetables are important components of a healthy diet but may serve as vehicles for transmission of intestinal parasites when contaminated during production, harvesting, transport, and marketing. This study assessed the prevalence and distribution of parasitic contamination in fresh vegetables cultivated on small-scale farms and marketed through wholesale and retail outlets in Hosanna town and surrounding areas. A cross-sectional study was conducted from December 2024 to June 2025 on 492 vegetable samples, including Ethiopian kale, Swiss chard, carrot, tomato, cabbage, and beetroot, collected from small-scale farms (n = 96), wholesale markets (n = 216), and retail markets (n = 180). Samples were washed with physiological saline and microscopically inspected for intestinal parasite infectious stages. The study revealed that parasitic contamination increased significantly along the farm (preharvest)-to-market (postharvest) chain (p < 0.05), with 35.42% of preharvest farm samples, 46.30% of wholesale, and 72.78% of retail market (postharvest) samples testing positive. Among farm (preharvest) samples, Ethiopian kale (54.16%) and beetroot (41.66%) had the highest contamination rates, predominantly with Strongyloides stercoralis (33.33%) and Entamoeba histolytica/dispar (16.67%). In wholesale markets (postharvest), carrot showed the highest contamination (69.44%), whereas E. histolytica/dispar was the most frequently detected parasite (30.56%). Retail markets (postharvest) exhibited the greatest contamination burden, particularly tomatoes and cabbages from Arada and Gofer Meda markets, where E. histolytica/dispar reached 50.00%. Leafy and root vegetables, especially Ethiopian kale and carrots, were the most contaminated with parasites. Fresh vegetables grown and sold in the study area were commonly contaminated with intestinal parasites, with retail vegetables showing the highest contamination rates. Enhanced hygiene practices during production, handling, and marketing, alongside targeted public health interventions, are crucial to reducing foodborne parasitic infections in the community.