Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

The Tapeworm Hymenolepis diminuta as an Important Model Organism in the Experimental Parasitology of the 21st Century.

The tapeworm Hymenolepis diminuta is a common parasite of the small intestine in rodents but it can also infect humans. Due to its characteristics and ease of maintenance in the laboratory, H. diminuta is also an important model species in studies of cestodiasis, including the search for new drugs, treatments, diagnostics and biochemical processes, as well as its host-parasite interrelationships. A great deal of attention has been devoted to the immune response caused by H. diminuta in the host, and several studies indicate that infection with H. diminuta can reduce the severity of concomitant disease. Here, we present a critical review of the experimental research conducted with the use of H. diminuta as a model organism for over more than two decades (in the 21st century). The present review evaluates the tapeworm H. diminuta as a model organism for studying the molecular biology, biochemistry and immunology aspects of parasitology, as well as certain clinical applications. It also systematizes the latest research on this species. Its findings may contribute to a better understanding of the biology of tapeworms and their adaptation to parasitism, including complex correlations between H. diminuta and invertebrate and vertebrate hosts. It places particular emphasis on its value for the further development of modern experimental parasitology.

Infection with intestinal helminth (Hymenolepis diminuta) impacts exploratory behavior and cognitive processes in rats by changing the central level of neurotransmitters.

Parasites may significantly affect the functioning of the host organism including immune response and gut-brain-axis ultimately leading to alteration of the host behavior. The impact of intestinal worms on the host central nervous system (CNS) remains unexplored. The aim of this study was to evaluate the effect of intestinal infection by the tapeworm Hymenolepis diminuta on behavior and functions of the CNS in rats. The 3 months old animals were infected, and the effects on anxiety, exploration, sensorimotor skills and learning processes were assessed at 18 months in Open Field (OF), Novel Object Recognition (NOR) and the Water Maze (WM) tests. After completing the behavioral studies, both infected and non-infected rats were sacrificed, and the collected tissues were subjected to biochemical analysis. The levels of neurotransmitters, their metabolites and amino acids in selected structures of the CNS were determined by HPLC. In addition, the gene expression profile of the pro- and anti-inflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-10) was evaluated by Real-Time PCR to determine the immune response within the CNS to the tapeworm infection. The parasites caused significant changes in exploratory behavior, most notably, a reduction of velocity and total distance moved in the OF test; the infected rats exhibited decreased frequency in the central zone, which may indicate a higher level of anxiety. Additionally, parasite infestation improved spatial memory, assessed in the WM test, and recognition of new objects. These changes are related to the identified reduction in noradrenaline level in the CNS structures and less pronounced changes in striatal serotonergic neurotransmission. H. diminuta infestation was also found to cause a significant reduction of hippocampal expression of IL-6. Our results provide new data for further research on brain function during parasitic infections especially in relation to helminths and diseases in which noradrenergic system may play an important role.

Selection of new diagnostic markers for Dirofilaria repens infections with the use of phage display technology.

Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Effect of ocular demodicosis on the stability of the tear film and the tear break up time.

The aim of the study was to analyze the correlation between the presence of Demodex mites in the hair follicles of patients' eyelashes and the stability and break up time of the tear film assessed with the Non-Invasive Tear Break Up Times (NIBUT) method. 319 patients were included in the study (195 women, 124 men). The patients were divided into two groups: those with Demodex infestation and without visible symptoms of eyelid or eye surface diseases, and asymptomatic non-infested patients. The NIBUT analysis was performed with a 5 M keratograph (oculus). Non-invasive tests were performed to identify the first and mean values of the tear break up time. The first and mean tear break up time in the Demodex-infested group was lower than in the non-infested subjects. The difference was a highly statistically significant. There was a significant correlation with the age of the patients for the first break up time. The first break up time in both eyes decreased with the age of the Demodex-infested and non-infested patients. The NIBUT analyses indicate the impact of Demodex mites on the tear film stability. This may suggest possible association of demodicosis with dry eye syndrome.

The New Status of Parasitic Diseases in the COVID-19 Pandemic-Risk Factors or Protective Agents?

It is possible that parasites may influence the course of COVID-19 infection, as either risk factors or protective agents; as such, the current coronavirus pandemic may affect the diagnosis and prevention of parasitic disease, and its elimination programs. The present review highlights the similarity between the symptoms of human parasitoses and those of COVID-19 and discuss their mutual influence. The study evaluated selected human parasitoses with similar symptoms to COVID-19 and examined their potential influence on SARS-CoV-2 virus invasion. The available data suggest that at least several human parasitoses could result in misdiagnosis of COVID-19. Some disorders, such as malaria, schistosomiasis and soil-transmitted helminths, can increase the risk of severe infection with COVID-19. It is also suggested that recovery from parasitic disease can enhance the immune system and protect from COVID-19 infection. In addition, the COVID-19 pandemic has affected parasitic disease elimination programs in endemic regions and influenced the number of diagnoses of human parasitoses.

Immunoproteomic Analysis of Dirofilaria repens Microfilariae and Adult Parasite Stages.

Dirofilariarepens is a parasitic nematode causing a vector-borne zoonotic infection (dirofilariosis), considered an emerging problem in human and veterinary medicine. Currently, diagnosis is based on the detection of the adult parasite and microfilariae in the host tissues. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable and affordable serological diagnostic method is needed. Better characteristic of the parasite biology and its interaction with host immune system should help to achieve this goal. This study analyzes adult and microfilariae proteomes, and the use of one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) proteomics, immunoproteomics, and LC-MS/MS mass spectrometry allowed us to identify 316 potentially immunogenic proteins (75 belong to adult stage, 183 to microfilariae, and 58 are common for both). Classified by their ontology, the proteins showed important similarities and differences between both parasite stages. The most frequently identified proteins are structural, metabolic, and heat shock proteins. Additionally, real-time PCR analysis of some immunogenic targets revealed significant differences between microfilariae and adult life stages. We indicated molecules involved in parasite-host interactions and discussed their importance in parasite biology, which may help to reveal potential diagnostic antigens or select drug and vaccine targets.

Sparganosis (Spirometra) in Europe in the Molecular Era.

Sparganosis is a relatively neglected foodborne and waterborne disease caused by species of the tapeworm genus Spirometra, the global distribution of which has not been sufficiently recognized. Known mainly as a zoonosis of East Asia, its species are native to all inhabited continents including Europe. Spirometra has been reported from numerous wildlife species from 17 European countries, and a critical review confirmed 17 autochthonous and 8 imported human clinical cases. We present the first molecular evidence of the coincident presence of 2 species in Europe and review the current distribution to raise awareness of the parasite in this region. Spirometra erinaceieuropaei is restricted to Europe and Spirometra mansoni represents a lineage distributed mainly across Asia and Oceania that reaches Europe. The parasite is common in Eastern Europe and its distribution has potential to expand along with its invasive or migrating mammal hosts, spreading the risks of human infection.

Author Correction: Hybrid de novo whole-genome assembly and annotation of the model tapeworm Hymenolepis diminuta.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hybrid de novo whole-genome assembly and annotation of the model tapeworm Hymenolepis diminuta.

Despite the use of Hymenolepis diminuta as a model organism in experimental parasitology, a full genome description has not yet been published. Here we present a hybrid de novo genome assembly based on complementary sequencing technologies and methods. The combination of Illumina paired-end, Illumina mate-pair and Oxford Nanopore Technology reads greatly improved the assembly of the H. diminuta genome. Our results indicate that the hybrid sequencing approach is the method of choice for obtaining high-quality data. The final genome assembly is 177 Mbp with contig N50 size of 75 kbp and a scaffold N50 size of 2.3 Mbp. We obtained one of the most complete cestode genome assemblies and annotated 15,169 potential protein-coding genes. The obtained data may help explain cestode gene function and better clarify the evolution of its gene families, and thus the adaptive features evolved during millennia of co-evolution with their hosts.

Immunoproteomics and Surfaceomics of the Adult Tapeworm Hymenolepis diminuta.

In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.

Identification of immunogenic proteins of the cysticercoid of Hymenolepis diminuta.

BACKGROUND: A wide range of molecules are used by tapeworm metacestodes to establish successful infection in the hostile environment of the host. Reports indicating the proteins in the cestode-host interactions are limited predominantly to taeniids, with no previous data available for non-taeniid species. A non-taeniid, Hymenolepis diminuta, represents one of the most important model species in cestode biology and exhibits an exceptional developmental plasticity in its life-cycle, which involves two phylogenetically distant hosts, arthropod and vertebrate. RESULTS: We identified H. diminuta cysticercoid proteins that were recognized by sera of H. diminuta-infected rats using two-dimensional gel electrophoresis (2DE), 2D-immunoblotting, and LC-MS/MS mass spectrometry. Proteomic analysis of 42 antigenic spots revealed 70 proteins. The largest number belonged to structural proteins and to the heat-shock protein (HSP) family. These results show a number of the antigenic proteins of the cysticercoid stage, which were present already in the insect host prior to contact with the mammal host. These are the first parasite antigens that the mammal host encounters after the infection, therefore they may represent some of the molecules important in host-parasite interactions at the early stage of infection. CONCLUSIONS: These results could help in understanding how H. diminuta and other cestodes adapt to their diverse and complex parasitic life-cycles and show universal molecules used among diverse groups of cestodes to escape the host response to infection.

New Data on Human Macrophages Polarization by Hymenolepis diminuta Tapeworm-An In Vitro Study.

Helminths and their products can suppress the host immune response to escape host defense mechanisms and establish chronic infections. Current studies indicate that macrophages play a key role in the immune response to pathogen invasion. They can be polarized into two distinct phenotypes: M1 and M2. The present paper examines the impact of the adult Hymenolepis diminuta (HD) tapeworm and its excretory/secretory products (ESP) on THP-1 macrophages. Monocytes were differentiated into macrophages and cultured with a living parasite or its ESP. Our findings indicate that HD and ESP have a considerable impact on human THP-1 macrophages. Macrophages treated with parasite ESP (with or without LPS) demonstrated reduced expression of cytokines (i.e., IL-1α, TNFα, TGFß, IL-10) and chemokines (i.e., IL-8, MIP-1α, RANTES, and IL-1ra), while s-ICAM and CxCL10 expression rose after ESP stimulation. In addition, inflammatory factor expression rose significantly when macrophages were exposed to living parasites. Regarding induced and repressed pathways, significant differences were found between HD and ESP concerning their influence on the phosphorylation of ERK1/2, STAT2, STAT3, AMPKα1, Akt 1/2/3 S473, Hsp60, and Hck. The superior immunosuppressive properties of ESP compared to HD were demonstrated with lower levels of IL-1ß, TNF-α, IL-6, IL-23, and IL-12p70 following stimulation. The presence of HD and its ESP were found to stimulate mixed M1/M2 macrophage phenotypes. Our findings indicate new molecular mechanisms involved in the response of human macrophages to tapeworm infection, this could be a valuable tool in understanding the mechanisms underlying the processes of immune regulation during cestodiasis.

Comparative Proteomic Analysis of Hymenolepis diminuta Cysticercoid and Adult Stages.

Cestodiases are common parasitic diseases of animals and humans. As cestodes have complex lifecycles, hexacanth larvae, metacestodes (including cysticercoids), and adults produce proteins allowing them to establish invasion and to survive in the hostile environment of the host. Hymenolepis diminuta is the most commonly used model cestode in experimental parasitology. The aims of the present study were to perform a comparative proteomic analysis of two consecutive developmental stages of H. diminuta (cysticercoid and adult) and to distinguish proteins which might be characteristic for each of the stages from those shared by both stages. Somatic proteins of H. diminuta were isolated from 6-week-old cysticercoids and adult tapeworms. Cysticercoids were obtained from experimentally infected beetles, Tenebrio molitor, whereas adult worms were collected from experimentally infected rats. Proteins were separated by GeLC-MS/MS (one dimensional gel electrophoresis coupled with liquid chromatography and tandem mass spectrometry). Additionally protein samples were digested in-liquid and identified by LC-MS/MS. The identified proteins were classified according to molecular function, cellular components and biological processes. Our study showed a number of differences and similarities in the protein profiles of cysticercoids and adults; 233 cysticercoid and 182 adult proteins were identified. From these proteins, 131 were present only in the cysticercoid and 80 only in the adult stage samples. Both developmental stages shared 102 proteins; among which six represented immunomodulators and one is a potential drug target. In-liquid digestion and LC-MS/MS complemented and confirmed some of the GeLC-MS/MS identifications. Possible roles and functions of proteins identified with both proteomic approaches are discussed.

Do Demodex mites play a role in pterygium development?

Numerous reports point to Demodex mites as the aetiology of certain eye and skin conditions, hence it is highly probable that infestation with these mites may also play a role in the aetiology and pathogenesis of pterygium. Therefore here we present a hypothesis: whether the presence of Demodex mites in eyelash hair follicles significantly correlates with the presence of pterygia, which could point to a potential role of these mites in the development of such lesions. We present preliminary data for supporting this hypothesis. These results were collected from a group of 69 subjects, including 39 with pterygium. Subjects were studied for the presence of Demodex folliculorum and/or D. brevis within eyelash follicles. The sample was defined as positive if at least one parasite, larva or egg were present. Preliminary statistical analyses were performed, and indicated a significant relationship between Demodex mite infestation and the presence of pterygium (p<0.05). This suggests that demodicosis could be one of the factors causing pterygium and significantly contribute to its development. Subjects who had both pterygium and Demodex infestation formed a dominant group. The proportion of subjects with both pterygia and Demodex presence is high at 93.33%, while the proportion of subjects without pterygia who have mites is low at 20.51%. Aforementioned results support our working hypothesis that infestation with Demodex mites may result in pterygium development and therefore patients diagnosed with pterygium should be assessed for the presence of Demodex to verify our preliminary results. In the present paper, we discuss potential influence of Demodex on conjunctiva and propose a hypothetical pathological mechanism linking the development of pterygia with demodicosis.

Mass spectrometry analysis of the excretory-secretory (E-S) products of the model cestode Hymenolepis diminut a reveals their immunogenic properties and the presence of new E-S proteins in cestodes.

Hymenolepis diminuta is an important model species in studies of therapeutics, biochemical processes, immune responses and other aspects of cestodiasis. The parasite produces numerous excretory-secretory (E-S) proteins and a glycocalyx covering its body. Our study focused on the mass spectrometry analysis of the E-S material with an objective to determine if E-S contains any new proteins, in particular those that can be identified as: antigens, vaccine candidates and drug targets. These proteins might engage directly in host-parasite interactions. Adult parasites collected from experimentally infected rats were cultured in vitro for 5 and 18h. Immunoblotting was used to verify which E-S protein bands separated in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) react with specific antibodies from sera of infected rats. We identified thirty-nine proteins by LC-MS/MS (liquid chromatography mass spectrometry). Results indicated the presence of proteins that have never been identified in cestode E-S material. Immunoblotting showed the immunogenicity of E-S products of H. diminuta, most probably associated with the presence of proteins known as antigens in other flatworm species. Among identified proteins are those engaged in immunomodulatory processes (eg. HSP), in response to oxidative stress (peroxidasin) or metabolism (eg. GAPDH). The predominant functions are associated with metabolism and catalytic activity. This is the first study identifying E-S-proteins in adult tapeworms, thus providing information for better understanding host-parasite interrelationships, and may point out potential targets for vaccines or drug discovery studies, as among the proteins observed in our study are those known to be antigens.

Ultrastructural study of vitellogenesis of Ligula intestinalis (Diphyllobothriidea) reveals the presence of cytoplasmic-like cell death in cestodes.

BACKGROUND: The tapeworm Ligula intestinalis (Diphyllobothriidea) is one of the most fascinating cestode parasites because it may cause parasitic castration of its second intermediate host, teleost freshwater fishes, due to inhibition of production of fish gonadotropic hormones. Large-sized (length up to 1 m) larvae called plerocercoids develop several months in the body cavity of freshwater fish and affect host behavior to facilitate transmission to the final host, a fish-eating bird. Vitellogenesis, i.e. formation of vitellocytes, is a key process in formation and nutrition of female gametes, oocytes in many flatworms, mainly parasitic Neodermata. The present study provides the first ultrastructural evidence in flatworms (Platyhelminthes) of the process that is interpreted as cytoplasmic-like cell death, i.e. a special case of programmed cell death (paraptosis) in vitellocytes of L. intestinalis. RESULTS: As molecular markers for paraptosis are not yet available, its identification was based on morphological criteria. Electron microscopy analyses revealed evident structural changes in vitellocytes associated with progressive cytoplasmatic vacuolation, swelling of the granular endoplasmic reticulum and mitochondria. In addition, the present study has shown that vitellocytes of L. intestinalis share numerous features in common with the members of other earliest evolved eucestodes. CONCLUSIONS: The present study indicates that paraptotic-like cell death may occur in parasitic flatworms (Neodermata). The presence of GER-bodies in mature vitellocytes indicates close relationship between the Diphyllobothriidea, Caryophyllidea and Spathebothriidea, which are considered as the earliest evolved groups of the Eucestoda. Beyond the general similarities, however, a number of differences exist between the morphology, chemical composition and amount of these inclusions which could be due to the variations in their embryonic development, life cycle strategies and definitive host groups.

Demodex mites as potential etiological factor in chalazion - a study in Poland.

The aim of the study was to investigate the presence of Demodex in the hair follicles of eyelashes and their potential participation in the aetiology of chalazion in patients in Poland. The study of the correlation between the presence of Demodex spp. and chalazion has never been performed in patients in Europe. There is, therefore, a justified necessity to check whether Demodex mites can be a potential risk factor in the development of chalazion in the European population. The samples were examined by light microscope, using standard parasitological methods. A positive result was assumed in the presence of Demodex spp. Demodex was detected in 91.67% of patients with a chalazion. The presence of Demodex was found in subjects from all examined age groups. The results of statistical analysis unambiguously determined the existence of an interrelationship between the presence of Demodex and chalazion. Our results clearly indicate the existence of a correlation between the occurrence of Demodex spp. and chalazion. Confirmation of the positive correlation between Demodex and chalazion in a European population provides further evidence for the pathogenic role of Demodex in the development of eye diseases.

Demodex sp. as a Potential Cause of the Abandonment of Soft Contact Lenses by Their Existing Users.

Demodex mites may be a potential etiological factor in the development of various eye and skin disorders. The aim of the study was to investigate the presence of Demodex in the hair follicles of eyelashes and their potential influence on abandoning soft contact lenses which had been previously well tolerated by their users. A group of 62 users of contact lenses (28 with emerging discomfort and 34 without discomfort) were examined. There is a need to check the existence of a relationship between D. folliculorum or/and D. brevis infestation and the emergence of intolerance to the presence of soft contact lenses. The removed lashes were examined under light microscopy, applying standard parasitological methods if demodicosis is suspected. A positive result was assumed if at least one adult stage, larva, protonymph/nymph, or egg of D. folliculorum and/or D. brevis was present. A positive correlation was observed between the presence of Demodex and intolerance to contact lenses by their existing users (p < 0.05), and Demodex sp. infections were observed in 92.86% of patients with intolerance to contact lenses. Our results provide further evidence for the pathogenic role played by the mites in the development of eye diseases.