Inverse surveillance seeks to identify potential pathogens before diseases emerge in populations. In this study, we conducted a molecular survey of eukaryotic vector-borne pathogens (VBPs) within ectoparasite species to anticipate disease outbreaks in animals and humans. We analysed fleas, lice, mites, ticks and triatomines collected from rodents, birds, carnivores and from the environment across Chile. After extracting genomic DNA, we employed an 18S rRNA gene amplicon-based sequencing strategy using Oxford Nanopore Technologies to characterize eukaryotic VBPs. Our findings revealed a narrow taxonomic range of microorganisms, with Hepatozoon as a well-supported taxon. A single sequence matching the genus Babesia was additionally confirmed via BLAST. The bioinformatic pipeline allowed the recovery of high-quality Hepatozoon consensus sequences, enabling robust phylogenetic and population genetic analyses and providing the first molecular record of Hepatozoon in triatomines and the first genetic detection of this parasite genus in trombiculid and macronyssid mites in the Americas. Three different Hepatozoon lineages were detected, which clustered with others found in rodents and reptiles. Analyses of molecular variance indicate that host identity may play a more influential role than geographic region in shaping the genetic differentiation of Hepatozoon lineages in Chile. Our study provides new data on ectoparasite–host–parasite associations, demonstrating the utility of nanopore metabarcoding data for exploring VBPs’ diversity, thereby expanding the known arthropod associations of the Hepatozoon genus and suggesting the role of these ectoparasites as its potential vectors. As some Hepatozoon species can cause animal disease, these findings constitute an early warning for veterinarians.

Cystic echinococcosis (CE) is a zoonotic disease of high public health and economic importance. The disease is caused by various tapeworm species belonging to the Echinococcus granulosus sensu lato species complex. Among these, E. granulosus sensu stricto (genotypes G1 and G3) is the most common cause of human CE worldwide. The aim of this study was to analyse the genetic diversity and phylogenetic relationships of G1 and G3 isolates from Armenia and Türkiye, where CE has long been recognized as a significant public health concern. A large portion of the mitochondrial genome (11 675 bp) was sequenced for 85 parasite samples: 47 from Türkiye and 38 from Armenia. Samples originated from humans, sheep and cattle. For a global phylogenetic analysis, sequences from previous studies were included, yielding 280 G1 sequences and 55 G3 sequences from various host species and countries worldwide. The main results of this work revealed (i) high genetic diversity of G1 in Armenia and Türkiye; (ii) high genetic diversity of G3 in Armenia, whereas only a few representatives of G3 were detected in Türkiye; (iii) the subdivision of isolates of G1 and G3 from Armenia and Türkiye into several haplogroups; (iv) the dispersion of G1 and G3 haplotypes from Armenia and Türkiye across different haplogroups in global phylogenetic networks; and (v) the diffusion routes of G3 isolates from Türkiye to Armenia, from Iran to Armenia and from Iran to Greece. These findings highlight the importance of mitogenome analysis for monitoring the spread of CE among animals and humans.

The latitudinal patterns of the phylogenetic, functional and numerical interaction specificity (PIS, FIS and NIS, respectively) of fleas and mammals in their interaction networks, from 4 biogeographic realms, were investigated at the intraspecific, interspecific and community levels to understand whether the spatial variation of interaction specificity followed the predictions of a latitudinal specialization gradient. In fleas, no latitudinal pattern of PIS was detected. Flea FIS varied with latitude in all realms at the intraspecific level and in 2 realms at the interspecific and community levels. Latitudinal trends in NIS were detected in all realms (except Neotropics) at the intraspecific level and in the Old World realms at both higher levels. In hosts, PIS varied with latitude in the Afrotropics and the Nearctic at the intraspecific level and in the Afrotropics at the interspecific and community levels. FIS demonstrated a latitudinal trend in the Nearctic at the intraspecific level and in the Palearctic at the community level. Relationships between latitude and NIS were found in all realms (except Neotropics) at the intraspecific level and in the Old World realms at the interspecific and community levels. The directions of the latitudinal pattern of the same specificity facet could be positive or negative. They differed between fleas and hosts, realms and organizational levels. The occurrence and direction of the latitudinal specialization gradient are explained by the combined actions of evolutionary, historical and ecological factors. Pooling data from regions with different biogeographical histories and landscape structures, for a macroecological analysis, may produce spurious results.

Hypersaline ecosystems host simplified yet highly structured food webs, in which Artemia plays a central ecological role, including serving as an intermediate host for several avian cestodes. Flamingolepis liguloides is among the most widespread parasites associated with Artemia worldwide; however, its presence in Italy has not been confirmed since early reports from Sardinia in the 1990s. Here, we provide the first documented evidence of F. liguloides infecting parthenogenetic Artemia in the hypersaline lagoon of Salina dei Monaci (Apulia, Italy). Host and parasite identities were confirmed through an integrative approach combining morphological analyses with molecular data, including newly generated 18S and 28S rDNA sequences, the latter providing novel molecular information for the parasite. Among the 254 brine shrimp examined, prevalence reached 93.3%, with a mean abundance of 7.3 ± 10.1 cysticercoids and up to 73 larvae in a single host. Parasite abundance was positively correlated with host body size, consistent with cumulative exposure and size-dependent feeding capacities. Cysticercoids were distributed within the host body, with the thorax harbouring the highest parasite loads, while cysticercoids became progressively more frequent in other body regions in larger individuals. The high infection levels observed, together with the presence of large flamingo aggregations in the lagoon, indicate a stable and active transmission cycle. This study fills an important geographic gap in the known distribution of F. liguloides and highlights the Salina dei Monaci as a promising natural system for future investigations on host–parasite dynamics in Mediterranean hypersaline environments.

The study aimed to characterize the species composition and compare helminth populations and communities in samples of helminths collected from marsh frogs in 3 localities in the city of Kyiv. Twenty helminth species were recorded in total, of which 8 were found in all 3 localities: the nematodes Cosmocerca ornata and Icosiella neglecta, and the trematodes Diplodiscus subclavatus, Haematoloechus asper, Opisthioglyphe ranae, Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus. In helminth infracommunities, species richness and abundance were the highest in frogs from Olzhyn Island (median 7 and 98, respectively). Helminth species richness was the lowest in the infracommunities from Lake Tiahle (median 4), while the abundance was the lowest in the infracommunities from Lake Telbin (median 51.5). From 3 to 4 species predominated in separate helminth component communities based on their high prevalence and abundance in the studied samples: D. subclavatus, I. neglecta, P. medians and P. confusus in Lake Telbin; Codonocephalus urniger, I. neglecta, O. ranae and P. confusus in Lake Tiahle; C. urniger, O. ranae and Tylodelphys excavata in Olzhyn Island. According to our observations, intense urbanization of Lake Telbin affects rather the abundance than the species richness in helminth communities of the marsh frog. On the other hand, the relatively low abundance of helminths in the infrapopulations from Lake Telbin resulted in comparatively higher diversity indices for the helminth component community in this locality. Moderate urbanization, on the contrary, results in the strong predominance of some helminth species and low values of the diversity indices.

One quarter of humans are infected with soil-transmitted helminths (STHs) worldwide. African green monkey (AGM, Chlorocebus aethiops sabaeus) interacts directly or indirectly with humans frequently on the island of St Kitts. They serve as a reservoir for zoonotic pathogens of significant public health concerns. We have previously demonstrated that AGMs are infected with zoonotic parasites Trichuris trichiura and Hymenolepis diminuta. Nevertheless, research on their STHs is limited. In this study, faeces were opportunistically collected from wild AGMs near residential areas between May 2020 and March 2022. They were examined for zoonotic STHs using direct smears (2 per sample) and molecular detection (PCR and DNA sequencing). The positive detection rates for whipworm were 60.0%, 60.0% for individual smears and 77.8% when both smears were combined. For hookworm, the rates were 13.3%, 6.7%, and 15.6%, respectively. Agreement between direct smear and molecular detection was 44.4% for whipworm and 88.9% for hookworm. The study further compared the sensitivity of direct smear, flotation and molecular detection, demonstrating that prolonged deep freezing (−80 °C) minimally alters egg morphology but markedly impairs egg flotation, whereas PCR and direct smear remain informative for frozen faecal samples. DNA sequencing of the internal transcribed spacers identified the hookworm as Ancylostoma duodenale and Necator americanus, the human hookworms, for the first time genetically in AGMs. These findings underscore the need to include AGMs as an integral component of One Health strategies aiming at eliminating STH morbidity in pre-school and school-age children on St Kitts and other Caribbean islands.

The taxonomy of wide distributed parasites across continents has rarely been evaluated using molecular data, and it remains unclear whether their apparent broad distributions reflect true cosmopolitan and monophyletic lineages. There is also the expectation that long-term parasite–host associations shape the evolution of parasitic lineages and should therefore be reflected in parasite taxonomy. Laelaps Koch, 1836 (Mesostigmata: Laelapidae) is an example of a widespread genus, reported from multiple continents, parasitizing Cricetidae and Muridae rodents. The aim of this study is to evaluate whether Laelaps is monophyletic and truly broadly distributed across continents. To this end, we generated new nuclear (18S-ITS1-5.8S-ITS2-28S) and mitochondrial (cytochrome oxidase I) sequences from 8 species of Laelaps, and 4 related laelapid mite species of the genera Androlaelaps Berlese, 1903, Gigantolaelaps Fonseca, 1939 and Mysolaelaps Fonseca, 1936 associated with cricetid rodents from Argentina. Those sequences were analysed together with sequences from laelapids parasitizing native South American cricetids and from South Africa available in GenBank. Phylogenetic analyses revealed that Laelaps is polyphyletic. Within Cricetidae, tribal host association appears to drive phylogenetic structure, with cricetid-associated Laelaps lineages recovered as more closely related to Gigantolaelaps and Mysolaelaps than to each other. Murid-associated Laelaps from South Africa were recovered as lineages separated from cricetid-associated Laelaps, suggesting that the deep divergence between Cricetidae and Muridae may also shape laelapid phylogeny, a pattern that requires broader sampling to be conclusively evaluated. These results challenge the monophyly and broad distributional status of Laelaps and highlight the need for integrative taxonomic revision of this genus.

The genus Kalicephalus Molin, 1861 comprises hookworms parasitizing the gastrointestinal tract of reptiles, particularly snakes, and currently includes 34 recognized species worldwide. Despite their diversity, the taxonomy of the genus remains poorly resolved because most species have been described primarily from morphology and molecular data remain limited. During a helminthological survey of monocled cobras (Naja kaouthia) from central Thailand, large hookworms were recovered from the oesophagus that corresponded morphologically to the group traditionally assigned to Occipitodontus but differed from previously described species. Using an integrative taxonomic approach combining light microscopy, scanning electron microscopy, morphometric analyses and molecular phylogenetic analyses based on nuclear 18S ribosomal RNA (18S rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) sequences, Kalicephalus colossus sp. nov. is described herein. The new species is distinguished by its exceptionally large body size, posteriorly projecting buccal capsule, equal alate spicules, lobate copulatory bursa and oesophageal localization. Multivariate analyses revealed clear sexual dimorphism without evidence of discrete morphotypes. Phylogenetic analyses recovered K. colossus sp. nov. as a distinct lineage within Kalicephalus, with genetic divergences of 1.1–1.4% for 18S rRNA and 12–13% for COI relative to congeners. The parasite was detected in 18% of examined hosts, with infection intensities ranging from 1 to 48 individuals per cobra, and was recorded only from suburban localities surrounding Bangkok. No obvious gross pathological lesions were observed during necropsy. The discovery of K. colossus sp. nov. expands current knowledge of reptile hookworm diversity in Southeast Asia and highlights the importance of integrative taxonomy for resolving parasite biodiversity.

Entamoeba bovis is a common intestinal amoeba of ruminants, yet molecularly confirmed data from Europe remain limited. We investigated the occurrence, morphology and genetic variability of E. bovis in Italian domestic and wild ruminants and developed a rapid species-specific polymerase chain reaction (PCR) assay. Faecal samples from calves (n = 45), goats (n = 26), sheep (n = 11) and Italian red deer Cervus elaphus italicus (n = 25) were examined by sediment microscopy and iodine staining. Amoebic stages were detected in 57% of bovine, 61.5% of caprine, 100% of ovine and 56% of red deer samples. Across hosts, cysts were consistently uninucleate and displayed typical E. bovis morphology, including frequent chromatoid bodies and an iodophilic glycogen mass. Representative positives were sequenced at the 18S rRNA locus (∼800 bp), confirming E. bovis (98.6–99.8% identity; 100% query coverage). Maximum-likelihood phylogenetic analysis placed all Italian isolates within the E. bovis clade and indicated a shared lineage across domestic ruminants and red deer, with no segregation by host or ecological setting. A newly designed E. bovis-specific PCR (375 bp) consistently yielded a single amplicon in all sequenced positives, supporting its use for rapid screening without sequencing.

During a parasitological survey of the cockscomb hydrobe, Littoridinops monroensis (Gastropoda: Cochliopidae), sympatric saltmarsh fishes and the American alligator, Alligator mississippiensis (Alligatoridae) within a saltmarsh pond in the Bon Secour Bay (Mobile Bay, Alabama, north-central Gulf of America) and other localities, we collected specimens of Proctocaecum diploporum (Stunkard, 1931) Baugh, 1957 and Proctocaecum coronarium (Cobbold, 1861) Brooks, 1980 (Cryptogonimidae). Fish scales were infected with 2 metacercarial cyst size classes, a small (<200 μm) and a large (>240 μm), suggesting the presence of distinct but closely related sympatric cryptogonimids. Morphological and nucleotide evidence demonstrated that P. diploporum (small cyst) and P. coronarium (large cyst), previously regarded as taxonomic synonyms, share the same first intermediate host (cockscomb hydrobe), are encysted within the scales of the same fish host species, and mature in the intestine of the same definitive host. The taxonomy of these trematodes and that of another similar American alligator trematode, Caimanicola pavida (Brooks and Overstreet, 1977) Brooks, 1980, needed reconsideration. Herein, we describe the first complete and naturally occurring life cycle of a reptile-infecting cryptogonimid, provide the first nucleotide data for a species of Proctocaecum, taxonomically reassess both P. diploporum and P. coronarium, and designate a neotype for P. coronarium. We consider C. pavida a junior subjective synonym of P. coronarium. Phylogenetically related and sympatric trematodes that use the same invertebrate and vertebrate hosts such as these Proctocaecum spp. could be a good model to further explore the mechanisms of speciation and host specificity among trematodes with complex life cycles.