UNASSIGNED: Medicinal leeches (Hirudo spp.) have been used for therapeutic purposes in humans since ancient times. Because of their growth conditions, leeches carry certain bacteria and endosymbionts (e.g., Aeromonas spp). In both leech farms and hirudotherapy clinics, there are no reliable antiseptics that can be used with leeches. This study aimed to determine whether methylene blue (MB) is a safe antiseptic for medicinal leeches and assess its safe usage.
UNASSIGNED: This study evaluated the efficacy of MB by determining lethal concentrations (LC), effective concentrations (EC), and lethal times (LT) for the medicinal leech Hirudo verbena Carena, 1820. A total of 570 H. verbana specimens obtained from a local farm were used in this study. Eighteen different concentrations of MB (between 1 ppm and 512 ppm) were tested.
UNASSIGNED: The LC50 and EC50 values for H. verbana were determined to be 60.381 (53.674-66.636) ppm and 2.013 (1.789-2.221) ppm, respectively. The LT50 durations for MB concentrations of 32 and 512 ppm were calculated as 212.92 h (138.43 h-1485.78 h) and 17.82 h (8.08 h-23.90 h), respectively.
UNASSIGNED: The results show that MB concentrations between 2 and 19 ppm can be safely used as antiseptics in hirudotherapy clinics and leech farms to address bacterial concerns caused by medicinal leeches.
UNASSIGNED: Tıbbi sülükler (Hirudo spp.) eski çağlardan beri insanlarda tedavi amaçlı kullanılmaktadır. Sülükler, büyüme koşulları nedeniyle bazı bakterileri ve endosimbiontları (örneğin; Aeromonas spp.) taşırlar. Hem sülük çiftliklerinde hem de hirudoterapi kliniklerinde sülüklerle birlikte kullanılabilecek güvenilir antiseptikler bulunmamaktadır. Bu çalışmanın amacı, metilen mavisinin (MB) tıbbi sülükler için güvenli bir antiseptik olup olmadığını belirlemek ve güvenli kullanımını değerlendirmektir.
UNASSIGNED: Bu çalışmada, tıbbi sülük Hirudo verbana Carena, 1820 için ölümcül konsantrasyonlar (LC), etkili konsantrasyonlar (EC) ve ölümcül süreler (LT) belirlenerek MB’nin etkinliği değerlendirilmiştir. Bu çalışmada yerel bir çiftlikten elde edilen toplam 570 H. verbana örneği kullanılmıştır. On sekiz farklı MB konsantrasyonu (1 ppm ile 512 ppm arasında) test edilmiştir.
UNASSIGNED: H. verbana için LC50 ve EC50 değerleri sırasıyla 60.381 (53.674-66.636) ppm ve 2.013 (1.789-2.221) ppm olarak belirlenmiştir. 32 ve 512 ppm MB konsantrasyonları için LT50 süreleri sırasıyla 212.92 saat (138.43 saat-1485.78 saat) ve 17.82 saat (8.08 saat-23.90 saat) olarak hesaplanmıştır.
UNASSIGNED: Sonuçlar, 2 ila 19 ppm arasındaki MB konsantrasyonlarının, tıbbi sülüklerin neden olduğu bakteriyel endişeleri gidermek için hirudoterapi kliniklerinde ve sülük çiftliklerinde antiseptik olarak güvenle kullanılabileceğini göstermektedir.

The use of medicinal leeches in clinical therapy has been employed for a long time, as it was originally recognized for exerting antithrombin effects. These effects were due to the ability of the leech to continuously suck blood while attached to human skin. According to Chinese Pharmacopoei, leeches used in traditional Chinese medicine mainly consist of Whitmania pigra Whitman, Hirudo nipponia Whitman, and Whitmania acranulata, but the latter two species are relatively scarce. The main constituents of leeches are protein and peptide macromolecules. They can be categorized into two categories based on their pharmacological effects. One group consists of active ingredients that directly target the coagulation system, such as hirudin, heparin, and histamine, which are widely known. The other group comprises protease inhibitor components like Decorsin and Hementin. Among these, hirudin secreted by the salivary glands of the leech is the most potent thrombin inhibitor and served as the sole remedy for preventing blood clotting until the discovery of heparin. Additionally, leeches play a significant role in various traditional Chinese medicine formulations. In recent decades, medicinal leeches have been applied in fields including anti-inflammatory treatment, cardiovascular disease management, antitumor treatment, and many other medical conditions. In this review, we present a comprehensive overview of the historical journey and medicinal applications of leeches in various medical conditions, emphasizing their pharmaceutical significance within traditional Chinese medicine. This review offers valuable insights for exploring additional therapeutic opportunities involving the use of leeches in various diseases and elucidating their underlying mechanisms for future research.

Leeches secrete various biologically active substances which have important medical and pharmaceutical values in antithrombotic treatments. Here, we provide a high quality genome of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis, based on which, we identified 22 antithrombotic gene families, including fourteen coagulation inhibitors, four platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. The total numbers of antithrombotic genes were similar between H. nipponia (N = 86) and H. tianjinensis (N = 83). Molecular evolution analysis showed that no significant differences were detected between the two species in any of the three selection indices (dN, dS, and dN/dS), nor in the number of sites under positive/purifying selection. RNA-Seq based gene expression analysis showed that the overall expression patterns of the antithrombotic gene families were not significantly deviated between the two species. Our results indicated that there were rather close similarities between the two leeches on genomic characteristics, especially for the molecular evolution and expression of antithrombotic genes. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from the two Asian medicinal leeches to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes of thrombosis.

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Nineteenth-century physicians increasingly favoured leeching - the placing of a live leech onto a patient\'s skin to stimulate or limit blood flow - as a cure for numerous ailments. As conviction in their therapeutic properties spread, leech therapy dominated European medicine; France imported over fifty million leeches in one year. Demand soon outpaced supply, spawning a lucrative global trade. Over-collection and farming eventually destroyed leech habitats, wreaked environmental havoc and forced European merchants to seek new supply sources. Vast colonies of leeches were found to inhabit the immense wetlands of the Ottoman Empire, which soon became a major exporter of medicinal leeches. Following the Treaty of Balta Liman (1838), the Ottoman state moved to exert control over the lucrative trade, imposing a tax on leech gathering and contracting with tax-farmers (mültezim) to collect the taxes. British diplomats, merchants and other stakeholders protested the imposition of the tax, as had previously happened with the commodification of wildlife; their pursuit of profit led collectors and farmers to over-gather leeches, with catastrophic consequences. By the end of the century, so great had their worth climbed that the leech population faced extinction. This paper situates medicinal leeches as therapeutic actors of history and adopts an interscale approach in formulating the human-leech interaction. It offers a substantive contribution to the history of medicine, in revealing the centrality of leeches to the rise of modern medicine and global trade, but also by making visible their role in shaping imperial diplomacy and worldwide economic markets.

Cationic antimicrobial peptides (CAMPs) are considered as next-generation antibiotics with a lower probability of developing bacterial resistance. In view of potential clinical use, studies on CAMP biocompatibility are important. This work aimed to evaluate the behavior of synthetic short CAMPs (designed using bioinformatic analysis of the medicinal leech genome and microbiome) in direct contact with blood cells and plasma. Eight CAMPs were included in the study. Hemolysis and lactate dehydrogenase assays showed that the potency to disrupt erythrocyte, neutrophil and mononuclear cell membranes descended in the order pept_1 > pept_3 ~ pept_5 > pept_2 ~ pept_4. Pept_3 caused both cell lysis and aggregation. Blood plasma and albumin inhibited the CAMP-induced hemolysis. The chemiluminescence method allowed the detection of pept_3-mediated neutrophil activation. In plasma coagulation assays, pept_3 prolonged the activated partial thromboplastin time (APTT) and prothrombin time (at 50 μM by 75% and 320%, respectively). Pept_3 was also capable of causing fibrinogen aggregation. Pept_6 prolonged APTT (at 50 μM by 115%). Pept_2 was found to combine higher bactericidal activity with lower effects on cells and coagulation. Our data emphasize the necessity of investigating CAMP interaction with plasma.

Plastics are a heterogeneous class of synthetic compounds that, due to their unique characteristics find numerous applications both in industrial and civil fields. However, despite the great advantages that these materials brought in everyday life, the plastic wastes resulting from their massive use represent one of the main environmental problems at the global level. Once released, plastics persist for a long time and are subjected both to biotic and abiotic processes leading to the formation of small particles, known as micro and to nanoplastics, that interact with organisms, accumulating inside tissues and risking to enter in the trophic chain. Among the different types of plastic, polypropylene (PP) is one of the diffused, widely exploited in food and textile industries for disposable packaging and to produce surgical masks. Owing to the huge distribution and the resultant abundant presence of PP waste products, it results necessary investigate the possible toxicity on living organisms. For these reasons, here we analyzed the effects of PP micro and nanoplastics dispersed in freshwater, using the medicinal leech Hirudo verbana as invertebrate model. To better follow the plastics fate, fluorescent particles, labeled with a fluorophore, have been used. Animals were examined at various timings after plastics exposure and results were analyzed by means of microscopy, immunofluorescent and molecular biology analyses. After assessing the entrance of PP fragments into leech tissues, the activation of the innate immune response was evaluated. The results show that the presence of micro and nanoplastics induces an initial physical protection that consists in the secretion of mucus, followed by an increase of blood vessels and the recruitment of immune cells, in particular macrophages. Moreover, macrophages were directly involved in both phagocytic and encapsulation processes, as demonstrated by acid phosphatase (ACP) histoenzymatic and Thioflavin-T assays, expressing specific pro-inflammatory factors, such as HvRNASET2 and HmAIF-1, as demonstrated by immunolocalization and qPCR experiments. Finally, the expression levels of genes related to oxidative stress-induced enzymes have been investigated, in order to evaluate the possible increase in reactive oxygen species (ROS), due to the entry into the leech tissues of PP micro and nanoplastics. This work allows deepening the current knowledge of the possible harmful effects on human health deriving from micro and nanoplastics dispersion, leading new insight about freshwater ecosystems that often represent the first environments interested in plastic pollution.

Blood sampling is a challenging procedure in many captive animals. Although manual restraint or anesthesia are usually possible, they entail intense stress and a high risk of injuries or organ failure. Blood sampling using medicinal leeches (Hirudo medicinalis) represents a promising non-invasive alternative to venipuncture; however, leech blood meal was to date used only for qualitative analyses such as genetic or serological screenings. Hence, the aim of this study was to evaluate the suitability of the leech blood sampling method for quantification of hematological and biochemical parameters. Medicinal leeches were manually applied on 67 zoo animals of eleven species, and control blood samples were obtained by venipuncture of the jugular vein. The leeches drew up to 20 ml of blood in 20 to 55 min. Although most hematological and biochemical parameters were significantly altered in leech-derived samples, their values showed strong (r = 0.62-0.79; 10/24 parameters) to very strong (r > 0.8; 13/24 parameters) correlations with venipuncture in all blood parameters, except for sodium (r = 0.39). As the parameter alterations and correlations were similar among species, simple cross-species regression formulas were sufficient to correct the alterations, thereby ensuring good repeatability between leeches and venipuncture in most parameters. Our data thus suggest that medicinal leeches can be used as a reliable non-invasive and stress-reducing alternative to standard venipuncture, even for quantitative assays. This opens new opportunities for a significant improvement to animal welfare in zoological gardens, conservation programmes, and ecophysiological research, where quantification of blood parameters is often needed.

Several studies have recently demonstrated that the correct regeneration of damaged tissues and the maintaining of homeostasis after wounds or injuries are tightly connected to different biological events, involving immune response, fibroplasia, and angiogenetic processes, in both vertebrates and invertebrates. In this context, our previous data demonstrated that the Hirudo verbana recombinant protein rHvRNASET2 not only plays a pivotal role in innate immune modulation, but is also able to activate resident fibroblasts leading to new collagen production, both in vivo and in vitro. Indeed, when injected in the leech body wall, which represents a consolidated invertebrate model for studying both immune response and tissue regeneration, HvRNASET2 induces macrophages recruitment, fibroplasia, and synthesis of new collagen. Based on this evidence, we evaluate the role of HvRNASET2 on muscle tissue regeneration and extracellular matrix (ECM) remodeling in rHvRNASET2-injected wounded leeches, compared to PBS-injected wounded leeches used as control. The results presented here not only confirms our previous evidence, reporting that HvRNASET2 leads to an increased collagen production, but also shows that an overexpression of this protein might influence the correct progress of muscle tissue regeneration. Moreover, due to its inhibitory effect on vasculogenesis and angiogenesis, HvRNASET2 apparently interfere with the recruitment of the myoendothelial vessel-associated precursor cells that in turn are responsible for muscle regeneration during wound healing repair.

Medicinal leech therapy promotes vascular flow and can be used to salvage grafts. Medicinal leeches have a symbiotic relationship with Aeromonas species and can therefore present a risk of bacterial transmission to patients. Antimicrobial prophylaxis is warranted for the duration of leech therapy, however, an institutional evaluation of 40 patients receiving medicinal leech therapy demonstrated poor adherence with recommendations. An electronic medical record order panel for antimicrobial prophylaxis with medicinal leech therapy was implemented, leading to a subsequent improvement in adherence to prophylaxis use, including significant increases in the ordering of antibiotics and the appropriate timing of initiation in the subsequent 10 patients receiving medicinal leech therapy after panel implementation. Aeromonas infections were rare before and after panel implementation, and developed only in the patient subset with non-optimized prophylaxis.