ul. Walerego Wróblewskiego 27B, 45-760 Opole

Leeches biology

CLASSIFICATION

Only the leeches belonging to Hirudo medicinalis species can be applied in medical treatment – hirudotherapy. Medical leeches Hirudo verbana bred in EURO-BION’S laboratory belong to this species.

To Hirudo medicinalis species belongs:

• Hirudo medicinalis (Europe)

• Hirudo verbana (Asia, Southern Europe)

• Hirudo nipponia (Japan)

Witmania pigra (China)

Hirudo septemstriata (Republic of Southern Africa)

Hirudo orientalis ( Iran, Uzbekistan)

Hirudo troctina ( Mediterranean Sea area)

Hirudo javanica (Bengal, Burma)

Hirudinaria manillensis (India, Malesia, Ceylon, Philipinnes )

Hirudinaria granulosa (India)

Haementeria ghilianii (Amazon river area)

Macrobdella decora (North America)

Philobdella gracile (North America coast)

Potamobdella oaxacensis (Mexico)

Limnobdella australis (Australia)

 

NATURAL  ENVIRONMENT

European medical leech can be primarily found in marshlands, oxbow lakes, backwaters, overgrown lakes and other freshwater pools in many countries all over the world.

ANATOMY

Its body is usually about 120 milimetres long and10 milimeters wide and has the ability to contract and extend. Intensive feeding over the span of 1.5 years allowed for raising a 44-centimeter specimen. Medical leeches have a dorsoventrally flattened body that bears no bristles, hence a sleek surface. The dorsal side is salient while the ventral side is flat. They may be colored yellow, green or dark brown. The ventral side of Hirudo Verbana is always one color – celadon or olive. The yellowish-orange streaks however, as well as bilateral yellowish-orange coloring of edges constitute their common features. Circular furrows divide body of the medical leech into repeating annuli. The external metamerism however does not match the internal one. Five external annuli correspond to one internal segment – boundaries between the actual segments of the body can be recognized by color. On the ventral side, segments are marked by small excretory orifices. There are however only 17 pairs of orifices for all 27 segments of the body. Blood of the medical leech has a red color.  At the anterior end of its body, leech has a sucker called the anterior sucker. At its bottom, there is an mouth opening that leads to the pharynx where three radially arranged jaws are located. Each jaw is equipped with 60 to 100 tiny chitinous teeth enabling the leech to make a skin incision that is approximately 1.5 millimeters deep. Three jaws leave on the skin a mark of an inverted Y (three radially arranged rays). Starting from the mouth opening is the muscular pharynx which extends from segments 4 through 8. Pharynx acts as a sucking apparatus and is surrounded by a single-cell glands. Their secretions (approximately one hundred have been identified) are transported with saliva to the bloodstream and tissues of a host (or a patient) within 10 centimeters from the site of bite. The mouth leads to a thin-walled crop which extends from segments 8 through 18. The crop is a container where the sucked blood is stored and does not perform any digestive functions. These are conducted by the next part of the digestive tract called the stomach. Its interior is covered with spiral plicae, or folds, which increase the absorption area. Flow of blood from the crop to the stomach proceeds slowly and digesting the entire content of the crop takes few months.   Starting from the stomach is the small intestine and a short straight intestine, that is rectum  which ends with anus. The anus is located on the dorsal side, just above the posterior sucker. Five pairs of eyes in the form of spots are located in the front of the dorsal side, extended from segments 2 through 6. They are very sensitive to the slightest changes in light intensity within 450 – 650 nm.

 

FEEDING

In natural habitat, medical leeches feed predominantly on blood of animals drinking at the waterholes such as frogs and other mammals. Mature adults can suck from 10 to 15 milliliters of blood during a single meal. Once satiated, they can live without feeding for as long as two years. Contrary to popular belief, leeches can attain maturity by feeding exclusively on blood of animals. In such cases though, they produce relatively less eggs and the whole process proceeds at a slower pace. Leeches are very lively and responsive to any stimuli generated by a potential host. Light sensitivity of the eye of medical leech. The electric response of a single light-sensitive eye cell at different wavelengths of light and constant light intensity. The maximal response (voltage) was recorded at the wavelength of 540 nm which corresponds to the green light. Medical leech is also responsive to temperature, smell, magnetic field, sounds, movements of water and air, gravity, electric field and constituents of chemical substances. Mechanoreceptors (sensory cells) distributed all over its skin are another sensory organs.

REPRODUCTION

Although medical leeches are hermaphrodites, they reproduce by cross-mating. In their natural habitat, leeches attain maturity after three years of living and copulate once a year, during the summer period. In laboratories where specific breeding conditions are maintained, Hirudo verbana can reproduce several times a year. Unpaired genital orifices are located on the ventral side of the body.

MEDICINAL LEECH IS NOWADAYS CONSIDERED A LIVING MINIATURE OF PHARMACOLOGICAL LABORATORY.

Major compounds produced by leeches:

HIRUDIN is the best known substance secreted by medical leech Hirudo medicinalis and at the same time, the best anti-clotting medication known to contemporary medicine. Hirudin is a non-enzymic polypeptide composed of 65 amino acids. Its exceptional properties are the result of high glutamine and asparagine content as well as its low isoelectric point. Hirudin stored as a lyophilisate at room temperature retains its properties for at least one year. In the water solution, protected with thymol crystals, it remains in effect for at least 6 months. Thermostability of hirudin rises as its pH decreases. It is quickly disintegrated by papain, pepsin and subtilopeptidase. Numerous active isoforms of hirudin have been identified.Hirudin is a thrombine inhibitor. Its effectiveness is a result of interaction with other secretions of salivary glands such as hementin, hementerin and antistatin. Leeches are also known to produce PSEUDOHIRUDIN which is composed of only 45 amino acids and bears no anti-coagulative properties (Baskova i Cherkesova 1980). Other secretions include HIRUDIN-PA, composed of 66 amino acids with a molecular mass 7026 and HIRUDIN VARIANT-2 PRECURSORbuilt of 72 amino acids with a molecular mass 7571. The effectiveness of hirudin is expressed using an international scale set by the National Institutes of Health (NIH), it reflects the anti-thrombine effectiveness of hirudin in At-U units. One anti-thrombine unit (At-U) corresponds to 0.1 microgram of pure hirudin. Each mature leech Hirudo medicinalis contains approximately 285 At-U of hirudin (Markwardt 1967), its concentration is however determined by the time that elapsed from the last feeding and was observed to reach the highest level on the 150th day (Zapkuvene and Sinyavichene 1976). In vivo research did not reveal any side effects caused by pure hirudin (Markwardt 1958, 1963; Markwardt, Hauptmann, Nowak, Klesson and Walsmann 1982). Intravnous injections of 20 milligrams of pure hirudin per kilogram of body weight performed on rabbits did not reveal the slightest degree of toxicity. White mice have been observed to survive intraperitoneal injections of pure hirudin in the dosage of one gram per kilogram of body weight. Hirudin is relatively quickly expelled from the bloodstream, 70 to 80 percents of the substance are excreted by kidneys within the first hour. For patients with certain blood related diseases, hirudin is considered to be a more effective anti-coagulant than the commonly applied polysaccharide heparin or dicoumarin, a medication composed of the clover extracts. It is especially so in patients with low levels or absence of anti-thrombine III and the related intravascular clots, renal failure, septic shocks or other specific hereditary conditions.

BDELIN is an inhibitor of proteases such as trypsin, chymotrypsin and plasmin which are responsible for inflammatory states and their diffusion within tissues. It can be found in at least two varieties: bdelin A and bdelin B. It is composed of 45 amino acids and has an affinity for the trypsin inhibitor produced in the pancreas of mammals. It is worth mentioning that bdelin and hirudin have exactly the opposite effect on blood clotting. Bdelin can be found in all body segments of the leech and in its salivary glands, but is mostly concentrated in the areas where reproductive organs are located.

HIRUSTASIN is yet another anti-coagulant produced by Hirudo medicinalis. Identified only recently, it is an organic compound classified to antistasins. It is an inhibitor of calicrein, trypsin, chymotrypsin and cathepsin G. It is composed of 55 amino acids and its molecular mass is 5878.

GILANTIN is a protein containing significant amounts of sulphur. It has been isolated from salivary glands of the Haementeria ghilianii leech. Gilantin, classified as antistasin, is an anticoagulant, a strong anti-metastatic medication and a very active inhibitor of factor Xa. It is composed of 119 amino acids and it molecular mass is 13317.

APYRASE is a powerful enzyme which improves fluidity of blood by reducing its viscosity.

EGLIN is a strong anti-inflammatory agent (inhibits factors responsible for inflammatory states) and a very active antioxidant. It is a potent inhibitor of proteases such as elastase and cathepsin G released by human polymorphonuclear leukocytes, which enables immediate localization of injuries and postoperative inlammations and their effective blocking. Eglins also degrade chymotrypsin and subtilisine – proteases produced by the Bacillus subtilis bacteria. A few varieties of eglins are produced by leeches, eglin C is the one most thoroughly studied. It has a molecular mass of  8100 and is composed of 70 amino acids, it is also relatively similar to the inhibitor of trypsin and chymotrypsin of potato and barley (27 identical amino acids at the minimum). Eglins belong to very durable compounds. Research provided evidence for their effectiveness in treatment of certain nervous tics and intestinal ulceration as well as the untypical protein disintegration related to septicaemia (Von Bary 1979; Seemiiller 1979).

HEMENTIN is a fibrinogenolitic enzyme capable of preventing blood clots and  disintegrating ones that already exist. It is produced in large amounts by both salivary glands of the Haementeria ghilianii leech, it operates in a different manner though, when compared to the antithrombine mechanisms of hirudin produced by Hirudo medicinalis. Hementin degrades fibrinogen and blood fibrin and is dissimilar to any anticoagulant produced by bloodsucking animals. Hementin is particularly interesting from the medical point of view for it remains non-responsive for the natural enzyme responsible for disintegrating blood proteins. Its molecular mass is 120.000. If stored at room temperature, hementin remains in effect for many hours losing its properties when exposed to the temperature of 60 degrees centigrade for over 15 minutes, or in contact with chelate compounds. From both salivary glands of Haementeria ghilianii about 4.5 milligrams of protein can be extracted, 2.8 percent of which is hementin. One leech contains amounts of hementin capable of incoagulating 300 milliliters of blood. It is still a subject of research, whether Hirudo medicinalis produces any significant amounts of this substance.

DESTABILASE is an enzyme characterized by strong anti-aggregative properties which allow for disintegration of clotted blood. This provides new opportunities for hirudotherapy. Destabilase also contains a unique low-molecular substance PROSTAGLANDIN which bears certain similarity to PROSTACYCLIN. Prostaglandin is responsible for regenerating blood vessels and digestive tract, maintaining them in good condition as well as regulating blood sugar concentration. The least examined property of this substance is its ability to ‘self-regulate’ blood pressure. Namely, if the blood pressure is too low, prostaglandin will automatically raise it to the optimal level and if its too high – it will reduce it. Attempts to explain this mechanism are constantly made and prostaglandin remains a subject of continuous interest among scientists.

HYALURINODASE is a substance which brings a double effect. It is a potent antibiotic and a factor enabling fast penetration of cell membranes within neighboring cells and tissues. In 1891, Heidenhain discovered a substance triggering such mechanism, but it was Claude (1937, 1940), later awarder with Nobel Prize, who conducted an in-depth research on the matter. In the course of a thorough study he observed that extracts from leeches have a strong loosening and dilating effect on human cells and tissues. In 1941 Hist for the first time demonstrated the potent antibiotic properties of this enzyme, his discovery however did not gain any recognition in the medical world at that time. An interesting feature of this enzyme is its ability to dissolve polysaccharide compounds which compose spore membranes of spores of numerous microorganisms. In the course of his experiments, Hist managed to effectively treat peritoneal infections in mice and guinea pigs by administering hyalurinodase. In 1969, Bagdasarova applied it as an effective treatment for glaucoma in people. Molecular mass of pure hyalurinodase is 28500. Under extreme pH conditions and at the temperature of 50 degrees centigrade, enzyme remains in effectfor one hour. Hyalurinodase retains its properties for the longest period of time if stored at the temperature of 38 degrees centigrade in an environment with pH factor 5.5.

LIPASES AND ESTERASES are substances which disintegrate lipids.

ANAESTHETIC SUBSTANCES are strong painkillers that still require precise identification. It is suspected that NEUROPEPTIDES produced by leeches and found attractive by human brain, trigger the analgesic effect. The argument behind this reasoning is the fact that these neuropeptides contain ENDORPHINES, commonly known as hormones of happiness. Aside from bringing pain relief, endorphines eliminate anxiety and euphoria bringing calmness, increase stress resistance, eliminate mental and physical addictions, bad mood, respiratory depression, nausea, depression and other symptoms observed in people addicted to drugs and alcohol. All leeches produce anaesthethic substances except for the Haemadipsa picta species, which have a sting and populate Borneo rainforests, and Haemadipsa ornata found among the climbers in the Indian bush. Bites of both are very painful (Moore 1927, 1929; Tracz 1982).

ANTIELASTASE reduces activity of elastases, hormones degrading skin elastin.

VASODILATOR is an organic compound very similar to histamine which still requires further research. In 1939 Lindemann described and explained dilation of blood vessels caused by secretion from salivary glands of Hirudo medicinalis leech. His research confirmed the previous observations (Magnus 1928) that in the area surrounding the site of a leech bite, capillary vessels dilate.

NEUROTRANSMITTERS are biochemical compounds regulating flow of electric impulses within nerve cells. Dopamine, serotonine, acetylcholine and encephalin are all neurotransmitters. They have a significant influence on reducing pain and changing behaviour by releasing anxiety or depression, trigerring sleep and inducing powerful emotional and sensory experiences without behavioral excitation, psychosis or paranoia that are caused by drugs like LSD or amphetamine. Moreover, serotonine plays a vital role in sensory perception and maintains body temperature on a proper level. It has been discovered that mental illnesses, primarily schizophrenia, may be caused by anomalies in serotonine transmission between neurons.

CHLOROMICETIN is a potent antibiotic with strong antibacterial properties. It is produced by a specific bacteria which lives in symbiosis with medical leech. Until recently Aeromonas hydrophila (= Pseudomonas hirudinis) was believed to be the symbiont. It still figures in specialist literature and numerous publications. In fact, application of state-of-the-art genetic technologies (biochemical tests and 16S rRNA sequencing) lead to a discovery that it is the Aeromonas veronii biovar sobria bacterium that is the actual symbiotic partner. The very same species of bacteria has been found in the digestive tract of vampire bats Desmodus rotundus that feed on blood and populate South America. Chloromicetin proved to be very effective in treating ulcers (Catterina 1897; Miihling 1899; Weiler 1949), relapsing fever (Weiler 1949), tetanus, meninigitis and infections caused by streptococcal bacteria (Steffen-hagen and Andrejew 1911; Hirst 1941; Weiler 1949 and others), tularaemia (Weiler 1949), erysipeloid in pigs (Weiler 1949) as well as infections caused by Staphylococcus aureus (Weiler 1949). The bacterial culture Aeromonas veronii biovar sobria in vitro kills tubercle bacilli (Schweizer 1936), eliminates dysentery and protects from diphteria and other diseases (Busing 1953).

ANTINEOPLASTIC SUBSTANCES. Scientific research conducted on mice with laboratorily injected tumor (T 241 sarcoma) proved these substances to inhibit development of neoplastic tumors. Introducing the extracts from salivary glands of Haementeria ghilianii to the treatment proved to trigger the tumor regression. The antineoplastic potential of leeches was for the first time suggested by Loeb and Fleischer in 1913 after they observed tumor regression in mice treated with leech extracts. Their findings however were ignored and very soon forgotten. As yet, this incredibly interesting substance, or substances, has not been identified.

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