Blastocystis sp. is a prevalent protistan parasite found globally in the gastrointestinal tract of humans and various animals. This review aims to elucidate the advancements in research on axenic isolation techniques for Blastocystis sp. and their diverse applications. Axenic isolation, involving the culture and isolation of Blastocystis sp. free from any other organisms, necessitates the application of specific media and a series of axenic treatment methods. These methods encompass antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined use of culture media. Critical factors influencing axenic isolation effectiveness include medium composition, culture temperature, medium characteristics, antibiotic type and dosage and the subtype (ST) of Blastocystis sp. Applications of axenic isolation encompass exploring pathogenicity, karyotype and ST analysis, immunoassay, characterization of surface chemical structure and lipid composition and understanding drug treatment effects. This review serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.

A total of 32 taxa of helminths were recovered from 52 individuals corresponding to 17 species of didelphiomorph marsupials collected across Bolivia. From these, 20 taxa are registered for the first time in this landlocked South American country, including the cestode Mathevotaenia bivittata, and the nematodes Moennigia sp., Travassostrongylus callis, Viannaia didelphis, V. hamata, V. metachirops, V. minispicula, V. philanderi, V. simplicispicula, V. skrjabini, V. viannai, Cruzia tentaculata, Monodelphoxyuris dollmeiri, Neohilgertia venusti, Pterygodermatites elegans, Pterygodermatites jeagerskioldi, Spirura guianensis, Gongylonemoides marsupialis, Turgida turgida and Trichuris reesali. We report for the first time parasites for Marmosops bishopi, Monodelphis emiliae, Monodeplhis glirina, Monodelphis sanctarosae, Monodelphis peruviana and Thylamys sponsorius and document 38 new records of parasites infecting marsupials. Twenty-six taxa of helminths infect 2 or more species of didelphiomorph marsupials, with the exception of Travassostrongylus callis, Viannaia didelphis, V. hamata, V. minispicula and V. hamate, which infected individuals of a single species.

Small nucleolar RNAs (snoRNAs) are short non-coding RNAs that are abundant in the nucleoli of eukaryotic cells and play a crucial role in various aspects of ribosomal RNA (rRNA) maturation, including modifications such as 2′-O-methylation or pseudouridylation. On the other hand, Giardia duodenalis is a microaerophilic, flagellated, binucleate protozoan responsible for causing giardiasis. Although numerous snoRNAs have been detected in Giardia, their investigation remains limited. Nevertheless, they have been found to play a crucial role in the rRNA precursor processing pathway and influence other cellular functions. In addition, it has been proposed that some microRNAs are generated from these snoRNAs through excision by the Giardia endoribonuclease Dicer. These microRNAs are believed to contribute to the regulation of antigenic variation, which allows the parasite to evade the host immune response. Specifically, they play a role in modulating variant-specific surface proteins (VSPs) and other cysteine-rich surface antigens (CSAs). The main objective of this study was to bring together the available data on snoRNAs in Giardia, uncovering their functions in various processes and their importance on a global scale. In addition, the research delved into potential microRNAs speculated to originate from snoRNAs, exploring their impact on cellular processes.

As a member of the Scathophagidae family, Scathophaga stercoraria (S. stercoraria) is widely distributed globally and is closely associated with animal feces. It is also a species of great interest to many scientific studies. However, its phylogenetic relationships are poorly understood. In this study, S. stercoraria was found in plateau pikas for the first time. The potential cause of its presence in the plateau pikas was discussed and it was speculated that the presence of S. stercoraria was related to the yak feces. In addition, 2 nuclear genes (18SrDNA and 28SrDNA), 1 mitochondrial gene (COI), and the complete mitochondrial genome of S. stercoraria were sequenced. Phylogenetic trees constructed based on 13 Protein coding genes (13PCGs), 18S and 28S rDNA showed that S. stercoraria is closely related to the Calliphoridae family; phylogenetic results based on COI suggest that within the family Scathophagidae, S. stercoraria is more closely related to the genus Leptopa, Micropselapha, Parallelomma and Americina. Divergence times estimated using the COI gene suggest that the divergence formation of the genus Scathophaga is closely related to changes in biogeographic scenarios and potentially driven by a combination of uplift of the Qinghai-Tibetan Plateau (QTP) and dramatic climate changes. These results provide valuable information for further studies on the phylogeny and differentiation of the Scathophaga genus in the future.

The island of Cyprus was a historical endemic area for cystic echinococcosis (CE) in the Mediterranean. During the last decades, Cyprus has been an open-air laboratory and a model for testing and implementing control measures aiming to eliminate CE as a public health problem. Despite control and surveillance measures implemented during last 50 years, molecular characterization of Echinococcus granulosus sensu lato specimens has been never provided. In February 2023, the carcass of a stray dog collected in the Nicosia district was examined by the Veterinary Services and found infected with Echinococcus spp. worms. The worms were sent to the European Union Reference Laboratory (EURLP) for species/genotype identification. The sequences analyses of nad2 and nad5 genes allowed us to identify the tapeworms as Echinococcus canadensis, genotype G7b. In November 2023, a parasitic liver cyst was observed during the post-mortem examination of a mouflon from the same area of the dog's finding. The cyst sample was also referred to EURLP for identification and comparison with tapeworms previously collected from the dog. The sequences analysis of cox1 gene allowed to identify the cyst as E. granulosus sensu stricto, genotype G1. The finding of 2 different species of E. granulosus s.l. in a limited area raises epidemiological questions on the origin of the samples: whether distinct transmission cycles are present or a recent introduction event have occurred. From a public health perspective, it will be essential to conduct further molecular epidemiology studies to clarify the recent transmission dynamics of Echinococcus species in Cyprus.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Ticks and tick-borne diseases affect humans, livestock, and wildlife in most regions of the globe. Although there are over 900 tick species globally, only approximately 10% of species are second to mosquitoes as major vectors of human and veterinary diseases. The 17 articles of this themed Special Issue highlight the current research trends associated with newly discovered tick species, concepts of tick evolution, new vaccinology approaches, factors affecting disease transmission, and factors affecting tick ecology and tick-borne disease epidemiology. Table 1 summarizes the articles in this Special Issue in alphabetical author order and Fig. 1 is a word cloud generated from the article titles. Of the 17 articles in this Special Issue, two are review articles (vaccinology) while the remaining 15 are original research articles. The topics range from tick control, to epidemiology, ecology, tick-borne disease control, tick-borne disease transmission, vaccine approaches, and the description of novel extant and extinct tick species. Fig. 2 is graphical representation of the articles within this Special Issue including tick hosts and the most representative tick species studied. The articles also include authors from most continents globally with first author contributions from Australia, Bangladesh, Brazil, Czech Republic, Germany, India, Mexico, Pakistan, South Africa, Spain, Turkey, United Arab Emirates, USA and Zambia. This issue is thus truly diverse which reflects the diversity of ticks, tick-borne diseases and they hosts they infest globally.

Identifying factors that drive among-individual variation in mixed parasitic infections is fundamental to understanding the ecology and evolution of host–parasite interactions. However, a lack of non-invasive diagnostic tools to quantify mixed infections has restricted their investigation for host populations in the wild. This study applied DNA metabarcoding on parasite larvae cultured from faecal samples to characterize mixed strongyle infections of 320 feral horses on Sable Island, Nova Scotia, Canada, in 2014 to test for the influence of host (age, sex and reproductive/social status) and environmental (location, local density and social group membership) factors on variation. Twenty-five strongyle species were identified, with individual infections ranging from 3 to 18 species with a mean richness (±1 s.d.) of 10.8 ± 3.1. Strongyle eggs shed in faeces were dominated by small strongyle (cyathostomins) species in young individuals, transitioning to large strongyles (Strongylus spp.) in adults. Egg counts were highest in young individuals and in the west or centre of the island for most species. Individuals in the same social group had similar parasite communities, supporting the hypothesis that shared environment may drive parasite assemblages. Other factors such as local horse density, sex, date and reproductive/social status had minimal impacts on infection patterns. This study demonstrates that mixed infections can be dynamic across host ontogeny and space and emphasizes the need to consider species-specific infection patterns when investigating mixed infections.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Investigations of the parasites associated with extinct avian species provide unique insights into the ecology and evolution of both hosts and their parasitic counterparts. In the present paper, a new quill mite species, Peristerophila conuropsis sp. n., belonging to the family Syringophilidae (Prostigmata: Cheyletoidea) is described from the Carolina parakeet Conuropsis carolinensis Linnaeus (Psittaciformes: Psittacidae). This new species was collected from museum dry skin of the Carolina parakeet, the only native representative of the Psittacidae in the United States, which was an abundant resident of the southeastern and midwestern states and has been extinct in the beginning of the 20th century. Comment on the current taxonomic state and host associations of the genus Peristerophila are provided. Based on the host associations and habitats occupied by Peristerophila and related genera on parrots, it is hypothesized with the high probability that P. conuropsis has been extinct along with its host.

Most studies concerning parasitic infections in European bison have been performed on free-ranging animals: comparatively little is known about the abomasal nematodes of captive wisents, which are widely used in reintroduction programmes. The aim of the study was to determine the infection level and species composition of abomasal nematodes in captive European bison in enclosures (including zoos) and breeding centres compared to free-ranging individuals. It also includes a morphological analysis of the parasites based on figures and measurement data. Altogether, 11 species of nematodes were detected, with both captive and free-ranging animals demonstrating similar species compositions. Among those, 2 species of blood-sucking nematodes were detected, including Ashworthius sidemi and Haemonchus contortus. Interestingly, A. sidemi was found in almost all free-roaming animals, but only in 1 captive European bison. In addition, H. contortus was predominant in captive animals. The morphological identification was confirmed molecularly for 5 nematode species: A. sidemi, H. contortus, Ostertagia kolchida, O. ostertagi and Spiculopteragia boehmi. The identification was performed using small subunit ribosomal rDNA. The study provides the first available set of specular lengths of the gastric nematodes of European bison, and the first molecular data of O. kolchida and S. boehmi derived from the same host species. Our findings may simplify the morphometrical and molecular identification of Trichostrongylidae species infecting European bison, and can be useful in developing new management strategies for populations of this near-threatened species in Europe.

During a 1-year study, Trichuris adults were obtained after necropsy of Arabian camels (Camelus dromedarius) from a slaughterhouse in Kuwait. Morphological and molecular identification was performed to confirm the identity of the Trichuris specimens obtained from C. dromedarius. Fifteen male Trichuris specimens were selected, and molecular identification was performed using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA, 16S ribosomal RNA genes and the nuclear internal transcribed spacer 2 (ITS2) region. Through phylogenetic analysis, 2 distinct groups were obtained using the mitochondrial genes, where group 1 showed a close relationship to Trichuris globulosa while group 2 showed a close relationship to Trichuris ovis, providing molecular evidence of a possible T. globulosa species complex. Additionally, the nuclear ITS2 region did not provide enough resolution to distinguish between the 2 groups of Trichuris specimens. Observation of morphological characters revealed variations in the shape of the male spicule sheath, where specimens present either a globular posteriorly truncated swelling or the absence of posteriorly truncated swelling. Moreover, the variations in male spicule sheath does not corroborate with the results of molecular data, suggesting the limited use of this character for identification of T. globulosa. In conclusion, molecular analysis suggests a possible species complex in T. globulosa, with the mitochondrial genetic markers successfully differentiating between the 2 groups. The limited use of the male spicule sheath as a diagnostic character for identification of T. globulosa is suggested.

Amphalius spirataenius belongs to Arthropoda, Insecta, Siphonaptera, Ceratophylloidea, Ceratophyllinae, Amphalius. Only 2 species from the subfamily Ceratophyllinae have been sequenced for mitogenomes to date. The genus Amphalius mitogenome research was still blank. The A. spirataenius mitogenome was determined, annotated and analysed for the first time in this study. The 14 825 bp long genome has the typical metazoan of 37 genes with insect ancestral genome arrangement pattern. There was no significant difference in codon usage of 13 protein-coding genes: UUA, UCU, GUU, ACU and GCU were the most frequently used codons. It was found that the reason for codon preference mainly contributed to natural selection base on PR2, ENC-plot and neutrality curve analysis. Evolutionary rate, conserved sites, variable sites and nucleotide diversity analysis indicated that nad6 of A. spirataenius had the fastest evolutionary rate, while cox1 had the slowest evolutionary rate. Phylogenetic trees were reconstructed based on 13 protein-coding genes and 2 rRNA genes datasets using Bayesian inference and maximum likelihood method. The phylogenetic tree supported that both Siphonaptera and Mecoptera were monophyletic, and were sister groups to each other. This study filled gap of the genus Amphalius mitogenome sequences and was of great significance for understanding evolution of the order Siphonaptera.

Manipulation of host behaviour by parasites to enhance transmission to the next host is a fascinating phenomenon that has interested scientists since the 1970s. It has been proposed that infection with the cestode Echinococcus multilocularis produces an impairment of the antipredatory behaviour in the rodent intermediate host common vole, Microtus arvalis, which may facilitate transmission of the tapeworm to the canid final host. In this study, we observed the behaviour of infected common voles at 12 weeks post-infection, when protoscoleces production and maturation commonly occurs, in order to assess behavioural changes compared to uninfected controls, that might ease predation in the wild. Infected and uninfected voles were monitored for 24 h to observe their spontaneous activity. In addition, the next day, both infected and uninfected voles were subjected to 4 different behavioural tests: open field test, barrier test, platform test and air-puff test in a running wheel. No significant difference between uninfected and infected voles emerged during the behavioural tests. However, observation of spontaneous activity revealed that infected voles increased their feeding frequency and spent significantly more time above bedding even when not eating, compared to the uninfected controls. In the wild, these behavioural changes increase the animals exposure to predators, raising their chance of becoming prey. These findings are the first direct evidence consistent with behavioural manipulation by E. multilocularis on common voles.

Mastomys natalensis and M. coucha are commensal rodent species endemic to Africa. A recent taxonomic revision within Mastomys leaves the parasite–host list of M. natalensis questionable and that of M. coucha incomplete. The current study aimed to develop a better understanding of the ectoparasite diversity associated with the 2 distinct but closely related rodent species and to explore the influence of host and habitat type on ectoparasite infestations. Between 2014 and 2020, 590 rodents were trapped in 3 habitat types (village, agriculture and natural) across a wildlife-human/domestic animal interface. In total 48 epifaunistic species (45 ectoparasitic and 3 predatory) represented by 29 genera from 4 taxonomic groups (fleas, lice, mites and ticks) were recorded. Only 50% of the epifauna were shared between the 2 rodent species, with mites the most speciose taxon in both host species. The abundance of epifaunistic individuals, and also those of mites and fleas, were significantly higher on male M. natalensis, while ticks were significantly higher on reproductively active M. natalensis. For both rodent species, infestations by most epifaunistic taxa (on M. natalensis) and some taxa (on M. coucha) were significantly lower in the village as opposed to the less disturbed agricultural and natural habitat types. The study highlights the importance of host life history, even in closely related rodent species, in shaping parasite profiles and a loss of parasite diversity in more extreme anthropogenic habitats.

Ticks are composed of 3 extant families (Argasidae, Ixodidae and Nuttalliellidae) and 2 extinct families (Deinocrotonidae and Khimairidae). The Nuttalliellidae possess one extant species (Nuttalliella namaqua) limited to the Afrotropic region. A basal relationship to the hard and soft tick families and its limited distribution suggested an origin for ticks in the Afrotropics. The Deinocrotonidae has been found in Burmese amber from Myanmar and Iberian amber from Spain, suggesting a wider distribution of the lineage composed of Deinocrotonidae and Nuttalliellidae. The current study describes 8 fossils from mid-Cretaceous (ca. 100 Ma) Burmese amber: 2 Deinocroton species (Deinocroton bicornis sp. nov.; Deinocroton lacrimus sp. nov.), 5 Nuttalliella species (Nuttalliella gratae sp. nov., Nuttalliella tuberculata sp. nov., Nuttalliella placaventrala sp. nov., Nuttalliella odyssea sp. nov., Nuttalliella tropicasylvae sp. nov.) and a new genus and species (Legionaris nov. gen., Legionaris robustus sp. nov.). The argument is advanced that Deinocroton do not warrant its own family, but forms part of the Nuttalliellidae comprising 3 genera, Deinocroton, Legionaris nov. gen. and Nuttalliella). Affinities of Burmese tick fossils to the Australasian region, specifically related to rifting of the Burma terrane from northern Australia ~150 million years ago, suggest that Nuttalliella had a much wider distribution than its current limited distribution. The distribution of Nuttalliella likely stretched from Africa over Antarctica and much of Australia, suggesting that extant members of this family may still be found in Australia. Considerations for the geographic origins of ticks conclude that an Afrotropic origin can as yet not be discarded.

Haemogregarine (Apicomplexa: Adeleorina) parasites are considered to be the most common and widespread haemoparasites in reptiles. The genus Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) can be found parasitizing a broad range of species and, in reptiles, they infect mainly peripheral blood erythrocytes. The present study detected and characterized a haemogregarine isolated from the lizard species, Ameiva ameiva, collected from the municipality of Capanema, Pará state, north Brazil. Blood smears and imprints from lungs, brain, heart, kidney, liver, bone marrow and spleen were observed using light microscopy and the parasite was genetically identified by molecular analysis. Morphological, morphometric and molecular data were obtained. Parasite gamonts were found in 49.5% (55/111) of the blood smears from A. ameiva, and were characterized as oval, averaging 12.0 ± 0.8 × 5.9 ± 0.6 μm2 in size, which displaced the nuclei of parasitized monocytes laterally. Parasite forms resembling immature gamonts were observed in the spleen and bone marrow of the lizards. Furthermore, phylogenetic analyses of 18S rRNA sequences did not reveal gene similarity with other Hepatozoon spp. sequences from reptiles. Thus, morphological and molecular analyses have identified a new species of Hepatozoon parasite, Hepatozoon lainsoni sp. nov., which infects monocytes of the A. ameiva lizard.

Toxoplasma gondii has at least 318 genotypes distributed worldwide, and tropical regions usually have greater genetic diversity. Campeche is a state located in the southeastern region of México and has favourable climate conditions for the replication and dissemination of this protozoan, similar to those in South American countries where broad genetic diversity has been described. Thus, in this study, 4 T. gondii isolates were obtained from tissues of stray dogs and free-range chickens in Campeche, México, and were genotyped by Mn-PCR-RFLP with 10 typing markers (SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) and 5 virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5) to provide new information about the distribution and virulence prediction of T. gondii genotypes. Two isolates of T. gondii genotype #116 and 2 of genotype #38 were obtained from stray dogs and chickens, respectively. The parasite load found in these species was between <50 and more than 35 000 tachyzoites per mg of tissue. Virulence marker genotyping revealed a recombinant 1&3 ROP5 RFLP pattern in 2 ToxoDB #116 isolates with no prediction of virulence in a murine model, while in the 2 ToxoDB #38 isolates, the ROP18/ROP5 combination predicted high virulence. Considering all the typed markers, there is a predominance of type I and III alleles, as constantly reported for the isolates characterized in various regions of México. It is crucial to determine their phenotype to corroborate the genetic virulence profile of the T. gondii isolates obtained in this study.

The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major. Bioinformatic analyses showed that the 4 distinctive extended winged-helix (eWH) domains and the coiled-coil motif are present in C82 in these microorganisms. Nevertheless, C82 in trypanosomatids presents certain unique traits, including an exclusive loop within the eWH1 domain. We found that C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. Knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of T. brucei. Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei, but not in L. major.