Alpha‐Gal/α‐Gal is an oligosaccharide produced by non‐primate mammals. Humans have developed an immune response mediated by anti‐α‐Gal antibodies that can trigger an allergic reaction and cause anaphylaxis. In recent years, cases of patients with delayed allergic reaction to mammalian meat have been reported worldwide. In Spain, these cases have been related to the species Ixodes ricinus L. (Ixodida: Ixodidae), whose distribution is located in the north of the country. In this work, the presence of α‐Gal in water‐soluble extracts from samples of salivary glands and digestive tracts of Hyalomma lusitanicum Koch (Ixodida: Ixodidae) both engorged and collected from vegetation were studied. The presence of that epitope was confirmed by the presence of reactive proteins of >250 kDa in both samples. The highest concentrations of α‐Gal were detected in salivary glands. Neither sex nor diet influenced the concentration of α‐Gal, which seems to indicate its endogenous production and its possible inoculation to the host during tick feeding.

Background/Objectives: This study investigates the potential of Anisakis sp. as a novel source of α-Gal (Galα1-3Galβ1-4GlcNAc-R) epitopes capable of inducing allergic sensitization in humans. While α-Gal is classically associated with delayed IgE-mediated hypersensitivity following tick bites, emerging evidence suggests that parasitic helminths such as Anisakis sp. may also express α-Gal-containing glycoconjugates, offering an alternative sensitization pathway. Methods: Protein extracts from Anisakis sp. third-stage larvae and mammalian tissues (beef, pork) were analyzed by SDS-PAGE and Western blot using a monoclonal anti-α-Gal antibody (clone M86), and α-Gal epitopes were detected by ELISA. Sera from urticaria patients, stratified by Anisakis sp. sensitization status, were evaluated for anti-α-Gal IgG, IgE, and IgG4 antibodies. Inhibition assays assessed cross-reactivity. Results: Results confirmed the presence of α-Gal epitopes on Anisakis sp. proteins, with prominent bands at ~250 kDa and 65 kDa. Urticaria patients sensitized to Anisakis sp. exhibited significantly elevated anti-α-Gal antibody levels compared to controls. Inhibition ELISA demonstrated substantial reduction in antibody binding with Anisakis sp. extracts, indicating shared antigenic determinants with mammalian α-Gal. Conclusions: These findings establish Anisakis sp. as a source of α-Gal-containing glycoproteins capable of eliciting specific antibody responses in humans, highlighting a potential parasitic route for α-Gal sensitization.

In extensive surveillance programmes of Culicoides biting midges (Diptera: Ceratopogonidae), morphological identification can be time‐consuming and difficult, while DNA barcoding, although highly accurate, may not be cost‐effective or suitable for rapid analysis, as it requires individual specimen processing. To address these limitations, we developed a rapid screening method using real‐time PCR assays with either SYBR Green or hydrolysis probe‐based detection chemistries. Species‐specific primers and, where necessary, hydrolysis probes were designed based on the updated sequences of the ITS2 region of seven Culicoides species. The specificity and efficiency of these assays were validated both in silico and through real‐time PCR testing on target and non‐target Culicoides species, tested individually and as mixed‐species samples. The new real‐time PCR assays detect the presence of key species including C. obsoletus , C. scoticus , C. chiopterus , C. dewulfi , C. pulicaris , C. punctatus and C. impunctatus in pools of specimens. The molecular techniques developed in this study provide a valuable tool for accurate and high‐throughput Culicoides surveillance, which can be used for year‐round monitoring of adult midges in traps, larvae and in environmental samples, potentially revealing novel insights into the spatial and temporal turnover of Culicoides species. These methods can be applied to large‐scale vector screening programmes involving pooled samples, addressing the limitations of previously described methods used in midge surveillance.

Culicoides biting midges are ectoparasites that act as vectors of animal and human pathogens. While much research effort has focused on species that serve as vectors for important livestock pathogens (belonging to the then Obsoletus and Pulicaris groups in the Palearctic region), other, less common, species are understudied. This study aimed to shed light on the genetic diversity of this lesser‐studied subgroup. A total of 133 Culicoides midges collected in Onderstepoort light traps in 13 sites across Ireland (June 2022–December 2023) were identified as Culicoides achrayi , Culicoides fascipennis , Culicoides festivipennis , Culicoides kibunensis , Culicoides impunctatus , Culicoides nubeculosus, Culicoides pallidicornis, Culicoides pictipennis , Culicoides poperinghensis , Culicoides salinarius and Culicoides vexans based on their wing morphology. Identities were confirmed using a PCR targeting a 478 bp fragment of the mitochondrial cytochrome C oxidase subunit gene (COI), followed by Sanger sequencing. Resulting sequences were compared against records on GenBank and aligned with international sequences to create phylogenetic trees and networks to assess their genetic diversity and relationship. Overall, 93.5% of morphological identifications were confirmed by COI barcoding. While several haplotypes were only recorded in Ireland, their apparent uniqueness is possibly a reflection of the limited number of published sequences in the database. Overall, there was limited evidence to suggest that midges collected in Ireland are genetically isolated from their continental European counterparts, with many sequences closely resembling or identical to those found elsewhere. The results suggests frequent exchanges between Irish and continental European midge populations, reinforcing the need for continued surveillance. Understanding the genetic structure of less‐well studied Culicoides species provides insights into midge population dynamics and connectivity, and informs future surveillance of midge‐borne pathogens in Ireland.