Hirudo Medicinalis Research Articles

Validation of Medicinal Leeches (Hirudo medicinalis) as a Non-invasive Blood Sampling Tool for Hematology and Biochemistry Profiling in Mammals.

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.

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients

BackgroundLeeches are classic annelids that have a huge diversity and are closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients.ResultsIn this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome size. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20 and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine patterns. Finally, we systematically explored and compared the active substances in the genomes of three leech species. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules.ConclusionsThis study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.

Approaches to studying injury-induced sensitization and the potential role of an endocannabinoid transmitter.

Endocannabinoids are traditionally thought to have an analgesic effect. However, it has been shown that while endocannabinoids can depress nociceptive signaling, they can also enhance non-nociceptive signaling. Therefore, endocannabinoids have the potential to contribute to non-nociceptive sensitization after an injury. Using Hirudo verbana (the medicinal leech), a model of injury-induced sensitization was developed in which a reproducible piercing injury was delivered to the posterior sucker of Hirudo. Injury-induced changes in the non-nociceptive threshold of Hirudo were determined through testing with Von Frey filaments and changes in the response to nociceptive stimuli were tested by measuring the latency to withdraw to a nociceptive thermal stimulus (Hargreaves apparatus). To test the potential role of endocannabinoids in mediating injury-induced sensitization, animals were injected with tetrahydrolipstatin (THL), which inhibits synthesis of the endocannabinoid transmitter 2-arachidonoylglycerol (2-AG). Following injury, a significant decrease in the non-nociceptive response threshold (consistent with non-nociceptive sensitization) and a significant decrease in the response latency to nociceptive stimulation (consistent with nociceptive sensitization) were observed. In animals injected with THL, a decrease in non-nociceptive sensitization in injured animals was observed, but no effect on nociceptive sensitization was observed.

Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman

Ethnopharmacological relevanceThe prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. Aim of the studyWe aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. Materials and methodsW. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. ResultsThe anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2–8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. ConclusionOur results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors.

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

The duration of rest and feeding greatly affects the re-breeding of ectoparasites: Hirudo verbana, Hirudo medicinalis and Hirudo orientalis.

Ectoparasitic leeches have many biologically active substances in their body, which are able to show various therapeutic effects, which makes them very relevant in the study. Among them, the most common are medicinal leeches: Hirudo verbana, Hirudo medicinalis and Hirudo orientalis. They are listed in the Red Book as a vulnerable species, so their population is mostly supported in biolabs. Therefore, the search for different methods of their preservation is relevant. The aim of the work was to test the effect of the duration of rest and feeding of animals for re- reproduction, which will increase their population. For the study, three experimental animal groups were formed: 1 control - the animals were fed a week after the first reproduction, re-reproduction occurred not earlier than 2 months; 2 experimental - selected animals that remained clitellum after the first reproduction and again sent to the peat-soil environment for reproduction without rest and feeding; 3 experimental - selected animals that remained clitellum after the first reproduction, fed after 1 week, after another 1-2 weeks again sent to the peat-soil medium for reproduction. As a result of the study, the restoration of the physiological state after the first dilution in the first and third groups was registered. In the second group, the animals are depleted as a result of defective offspring, mortality of them and their offspring.

DNA Barcoding: Molecular Identification and Phylogenetic Analysis of Pheretimoid Earthworm (Metaphire sp. and Amynthas sp.) Based on Mitochondrial Partial COI Gene from Sialkot, Pakistan.

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya). Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Emotional component of pain perception in the medicinal leech?

Crump et al. have provided a series of criteria to assess animal sentience that is focused on the perception of pain, which is known to have both sensory and emotional components. They also provide a qualitative scoring system to assess data that address the eight criteria and apply this paradigm to decapod crustaceans. The criteria laid out have the potential to be applied to other invertebrates typically thought to have sensory response to tissue damage, but no emotional component to pain perception.

Morphological parameters of spleen and thymus of the male rats on the basis of the hirudological influence of Hirudo verbana.

The aim of this work is to study the morphological parameters of the spleen and thymus of rats along with hirudological influence from Hirudo verbana. The work was carried out on 40 male outbred laboratory rats weighing 280-300 grams, age 6.5-7 months. Animals that had passed the quarantine regime and had no external manifestations of the disease were used for the study. Manipulations with animals were performed in compliance with regulated norms and rules for the treatment of laboratory animals. For the study, two groups of animals were formed - control and experimental. After fixation of rats, the medical leech Hirudo verbana with an average weight of 0.8 ± 0.1 g was put on the animals of experimental group. The place of setting is the coccyges zone. Four times with an interval of one time per week. Then the animals were decapitated and the spleen and thymus were removed for histological examination. The white pulp of the spleen was analyzed - the area and diameter of the lymphoid follicles and central arteries, the number of lymphocytes per unit area (400 μm2) in the lymphoid follicles, in the thymus - the number of cells per unit area, both in the cortex and in the medulla. Under the influence of biologically active substances of the medicinal leech in the white pulp of the spleen, the size of the lymphoid follicles and central arteries increases, the number of lymphocytes per unit area (400 μm2) of the lymphoid follicle increases, and in the thymus, both in the cortex and in the medulla, the number of cells increases per unit area (400 μm2), the predominance of the cortical over the medullar ones. As a result of the obtained experimental study, we revealed the immune stimulating effect of Hirudo verbana on the organs of the immune system of laboratory rats: thymus and spleen.

Effect of Methanolic Extract of Some Selected Plants on the Mortality of Leech (Hirudo medicinalis)

Effect of Methanolic Extract of Some Selected Plants on the Mortality of Leech (Hirudo medicinalis)

Eğirdir Gölü (Türkiye) çevresindeki sulak alanlarda yaşayan Akdeniz tıbbi sülüğü, Hirudo verbana popülasyonlarının büyüklüğü ve yapısı

In the present study size and structure of the Mediterranean medicinal leech (Hirudo verbana Carena, 1820) populations inhabiting wetlands around Lake Eğirdir (Turkey) were investigated. Population size was estimated by removal methods, age classes were estimated for the first time in leeches (Hirudinea) using “Modal Progression Analysis” of body length frequencies using Bhattacharya's method. The population size and biomass of medicinal leeches around Lake Eğirdir were estimated to be 1,562,696 ± 805,613 leeches and 467.26 ± 172.91 kg, respectively. Three age classes were identified; the age group of 0+ was dominant with 78.6%. While 89.9% of the individuals weighed less than 1 g, whereas the broodstock was estimated to represent only 7%. Body weight, body length and condition factor were found to be 0.52 ± 1.19 g, 4.5 ± 2.4 cm and 0.620 ± 0.123, respectively. This study reveals that the population size of H. verbana in the wetlands around Lake Eğirdir is relatively well preserved and there is no significant threat to the sustainability of the exploitation of the medicinal leech populations. It is concluded that the stability of the lake's water budget, the variety and abundance of the host in the undamaged wetlands, the socioeconomic structure of the leech collectors, the sales policies, and the effective activities of conservation and control units might affect conservation status of the medicinal leech populations inhabiting wetlands around Lake Eğirdir.

Influence of new antimicrobial peptides of the medicinal leech &lt;i&gt;Hirudo medicinalis&lt;/i&gt; on the functional activity of neutrophil granule proteins

BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms.&#x0D; AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins the main effector cells of the immune system.&#x0D; MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis.&#x0D; RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity.&#x0D; CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the bodys fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit if it necessary to protect against damage to the bodys own tissues.

Improving the Effectiveness of Therapeutic Measures for Generalized Periodontitis in Patients with Hypertension

The program of standard therapeutic measures in 45 patients with Generalized Chronic Periodontitis (GCP) and background hypertension, group I(a), included hirudotherapy, for comparison, the effectiveness of traditional treatment was evaluated in 40 patients, group I(b), who refused to use medical leeches. In the second comparison group (35 patients), patients with GCP, but normal blood pressure, also received only a set of traditional methods of treatment. The inclusion of hirudotherapy in the program of therapeutic measures in patients with GCP and hypertension makes it possible to optimize the onset of a persistent therapeutic effect, lengthen the period of remission of the pathological process in periodontal disease and significantly improves the quality of life of patients.

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients

Using the Helical Free Flap Based on the Retrograde Flow From Frontal Branch of Superficial Temporal Vessels in Lower Third Nasal Reconstruction.

Although the helical free flap is 1 of the most effective option for the lower third nasal reconstruction, the main limitation is still related to having a short pedicle length. In this report, the authors presented our experience in using the long pedicle helical free flap based on the frontal branch of the superficial temporal vessels in nasal reconstruction. Five patients, 3 alar full-thickness defects, 1 columellar defect, and 1 multiple subunit defect, underwent reconstruction using this technique. The pedicle length ranged from 4 to 6 cm. All the flaps were survived completely. However, vein congestion was seen post-operation in 1 case. This flap was saved by using medical leech. No vascular graft was needed. The helical free flap based on the retrograde flow of the frontal branch of the superficial temporal artery offers an option to overcome the limitation of short pedicle length. This composite flap is valuable for nasal alar and columellar reconstruction or even the larger defects, which involve multiple subunits.

Comparison of body wall histologic structure of two medicinal leeches Hirudo sulukii and Hirudo verbana (Hirudinida: Hirudinidae).

In this study, it was aimed to determine secretory cell types using histochemical properties of secretory cells and epidermis histology in the body wall of two medicinal leech species, Hirudo verbana and Hirudo sulukii. In addition, areas of epidermis epithelial cells, secretory cell types, and secretion areas of secretory cells stained with histochemical stains were statistically compared in both species. Epidermis is composed of single layer of cylindrical epithelium and secretory cells. The cuticle layer covers the epithelial layer. Some Type 1 cells within and close to the epidermis were determined as pear-shaped secretory cells. Type 2a and Type 2b secretory cells were found in large groups in the inner parts of the body wall, especially around muscles. While Type 1 cells were stained weakly with PAS and AB, Type 2b cells were stained darker than Type 2a cells. Statistical calculations showed that areas of epithelial and secretory cells were generally larger in H. sulukii than in H. verbana. Therefore, H. sulukii was thought to be a more resistant species compared to H. verbana. As secretion areas of secretory cells reacting with PAS and AB stains were generally larger in H. sulukii, it was concluded that mucus composition between the two species has different concentration.

Medical Leeches and Hirudotherapy: Where is Nurse’s Role?

Medical Leeches and Hirudotherapy: Where is Nurse’s Role?

Short tail stories: the hirudin-like factors HLF6 and HLF7 of the Asian medicinal leech, Hirudinaria manillensis

The leech-derived hirudins and hirudin-like factors (HLFs) share a common molecule structure: a short N-terminus, a central globular domain, and an elongated C-terminal tail. All parts are important for function. HLF6 and HLF7 were identified in the Asian medicinal leech, Hirudinaria manillensis. The genes of both factors encode putative splice variants that differ in length and composition of their respective C-terminal tails. In either case, the tails are considerably shorter compared to hirudins. Here we describe the functional analyses of the natural splice variants and of synthetic variants that comprise an altered N-terminus and/or a modified central globular domain. All natural splice variants of HLF6 and HLF7 display no detectable thrombin-inhibitory potency. In contrast, some synthetic variants effectively inhibit thrombin, even with tails as short as six amino acid residues in length. Our data indicate that size and composition of the C-terminal tail of hirudins and HLFs can vary in a great extent, yet the full protein may still retain the ability to inhibit thrombin.

Anaemia following medical leech therapy for caudal superficial epigastric skin flap salvage in a Shorthaired Chihuahua

AbstractA 6.5‐year‐old, 6.1 kg, spayed female Shorthaired Chihuahua was referred for surgical removal of a mast cell tumour regrowth on the right caudal thigh. In the right perineal region lateral to the base of the tail, there was a 4 × 5 cm soft tissue mass with focal alopecia in the centre. Complete excision of the mass was achieved with a caudal superficial epigastric skin flap for closure. At 5 days postoperatively, the distal 25% of the skin flap showed evidence of venous congestion. Medical leech therapy was performed, and the flap was salvaged successfully; however, the dog developed acute anaemia due to excessive blood loss and received a packed red blood cell transfusion. The dog recovered uneventfully after transfusion and the skin flap had healed completely at recheck (26 days postoperatively). To the authors' knowledge, this was the first report of anaemia requiring blood transfusion after leech therapy in dogs.

Spectral responses across a dorsal\u2013ventral array of dermal sensilla in the medicinal leech

How do animals use visual systems to extract specific features of a visual scene and respond appropriately? The medicinal leech, Hirudo verbana, is a predatory, quasi-amphibious annelid with a rich sensorium that is an excellent system in which to study how sensory cues are encoded, and how key features of visual images are mapped into the CNS. The leech visual system is broadly distributed over its entire body, consisting of five pairs of cephalic eyecups and seven segmentally iterated pairs of dermal sensilla in each mid-body segment. Leeches have been shown to respond behaviorally to both green and near ultraviolet light (UV, 365–375 nm). Here, we used electrophysiological techniques to show that spectral responses by dermal sensilla are mapped across the dorsal–ventral axis, such that the ventral sensilla respond strongly to UV light, while dorsal sensilla respond strongly to visible light, broadly tuned around green. These results establish how key features of visual information are initially encoded by spatial mapping of photo-response profiles of primary photoreceptors and provide insight into how these streams of information are presented to the CNS to inform behavioral responses.