Hirudotherapy in veterinary medicine

Hirudotherapy is the medical use of parasitic invertebrates: leeches, to treat wounded skins, due to their particular biological properties. These include phlebotomy (blood feeding off of host), and components of their complex saliva like anesthetic and anticoagulant substances. Although this non-invasive therapeutical use of leeches has been common in human medicine for a long time, it is also growing in veterinary medicine.

1 - Introduction & history

The symbiosis between leeches and their host has been established and used in medicine since a very long time ago.

The first trace of use are in 1600-1300 BC¹. In ancient Greece, medicinal philosophy proposed that a healthy body was based on the balance of 4humors (blood, phlegm, black- and yellow bile). Any sickness changing the skin colour to red or behaviour to sanguine, was thought to be caused by too much blood & the excess was removed by leeches. Bloodletting was also practiced in the Ancient Indian and Ancient Egyptian civilisation (trace of usage on a mural painting discovered in a tomb). Sushruta, the father of plastic surgery, wrote a text in 800 BC, called the Description of leech therapy. The Greek Nicander of Colophon (185-138 BC) and the Italian Themison of Laodicea (123-43 BC) recommended the use of leech for a therapeutical treatment.

In the medieval Europe,tonsillitis was treated by hanging a leech on a string, down into the patient’s throat.

In the years 1162-1231, Abd al-Latif al-Bahdadi, a physician and historian from Bagdad worked on hirudotherapy and that introduced modern use of the leeches.

It was in 1758 that Carolus Linnaeus studied and classified a European species: Hirudo medicinalis, one of the most therapeutically used in the present times. The other frequently used species is the East-asian Hirudo nipponia, classified later by Whitman in 1886. There are several other Hirudo species² of medicinal leeches, including H. orientalis (middle eastern and central asian), H. verbana (mediterranean), H. sulukii (turkish) and H. troctina (north-west african). Hirudinaria manillensis (mexican) and Macrobdella decora (north american) leaches are also used for medicinal means.

Broussais, a surgeon of the Napoleonic army, influenced a lot on the leech market, of which France became the biggest consumer. In the mid 1800s, the use of hirudotherapy reached such peak that supplies ran out. The intense demand led to some of the first legislative efforts. Leeches are now protected on national, european and international levels by the World Conservation Union (IUCN), the International Trade in Endangered Species (CITES), the Berne Convention and the European Union Habitat Directive.

The hirudotherapy trend then decreased in Europe until the end of the 19th century. Since the 1970s, the therapeutical use of leeches has gained back in popularity and is used throughout the world by doctors and surgeons, for tissue restoration andreconstructive microsurgery. It also has an increasing trend in veterinary medicine.

2 - General aspects of leeches

Leeches are worms that belong to the Annelida phylum, Clitellata class (have no parapodia nore setae), Arhynchobdellida order (presence of jaws but no proboscis) and the subclass Hirudinea.

a. Related anatomy & physiology

The body of theseannelids is cylindrical, dorso-ventrally flattened and divided into 33 or 34 segments, covered by an integument of epithelial, sensory and mucous glandular cells. Theses are covered by a thin cuticle, renewed when the leech moults. Their skin allows them to breath oxygen in water and in the atmosphere.

Members of the Hirudo family measure up to 20 cm in length when extended and can retract reducing their length by ⅔. This high contractility is due to the muscles fibres of the dermis, arranged in an external circular layer and an internal longitudinal layer. Therefore the 4 blood vessels composing the entire circulatory system are also contractile. Leech varieties are distinguishable due to their different pigmentations.

^{Fig. 1 - dorsal & ventral view of Hirudo verbana's body}

They have 5 pairs of eyes on the head, which seem to be light sensitive but they search for their host by the help of their olfactory system. They have two suckers, one at each end of the body. The posterior sucker is bigger and essential for locomotion. The anterior sucker consists of 3 monostichodont jaws and around a hundred teeth. The bite leaves a characteristic Y-shaped mark. The jaws contains some glands cells and small pores through which saliva and bioactive substances are released in the wound of the host.

• ^{Fig. 2 - anterior sucker of the leech & Fig. 3 - some morphologic specificites of leech}

1. Leech, 2. Anterior extremity magnified, 3. Jaw detached and magnified, 4. Part of belly magnified, 8. The jaw with its saw like tooth-plates, 9. The head with its three-parted month opening, 10. The upper side of the head with ten eyes. 11. Section of a cocoon with its eggs

The powerful pharynx evaginates into the wound to suck and swallow the blood. It also contains salivary glands. The 11 pairs of gastric caeca in the stomach have small valves allow them to store blood in large quantities, up to 10 times their body weight and therefore enable their fasting capacity; they can live up to a year without food. This is helped by their poikilothermic characteristic (their internal temperature varies constantly; the energy “saved” is used when movement is needed, like for feeding or reproduction).

Leeches arehermaphrodites. The male genital system contains 9 pairs of testicles, sperm ducts, epididymis, a prostate and an ejaculatory duct in a long filiform penis. The female genital system is formed of 2 ovaries and oviducts, a uterus and vagina. The eggs are placed in the clitellum; an enlarged glandular and non-segmented part of the body wall, situated near the head, which secretes a viscid sac. After one month, eggs are layed on land, in spring and summer, in cocoons containing each up to 30 youngs.

b. Saliva components

The saliva of leeches decreases the host’s humoral and immune response, which includes inflammation, swelling and pain.

Leeches secrete from the salivary gland a large variety of bioactive factors. These are released in the wound of the host while they are feeding themselves.

Hirudin is one of the components of the saliva that scientist have studied the most, it acts as an anticoagulant of the blood, inhibiting coagulation by binding to the thrombin molecule. “In 1950, Fritz Marquardt of Germany isolated a protein from H. medicinalis that he termed hirudin, and demonstrated its thrombin inhibitor properties. Hirudin is still regarded as the most potent natural inhibitor of thrombin, thanks to its high affinity for the protein”³.

The other important components of the saliva⁴ are :

• Hyaluronidase: it facilitate the diffusion of the pharmacological active substance in the tissues. This spreading factor also has an antibiotic effect.

• Calin:Inhibits coagulation by blocking the binding between the Von Willebrand factor and collagen. It also inhibits collagen-mediated platelets aggregation.

• Destabilase: thrombolytic effect and dissolves fibrin.

• Bdellin: has anti-inflammatory effect.Trypsin andplasmin are inhibited by it.

• Eglins: anti-inflammatory effect

• Hirustasin: inhibits kallikrein, chymotrypsin and trypsin.

• Anesthetic-like substances : reduce pain

• Histamine-like substances :act as vasodilator and increase the inflow of blood at the biting site

There are many other molecules (Chloromycetyn, Factor Xa inhibitor, complement inhibitors, acetylcholine, collagenase..) but with less effects. Recently the scientist found a new class of leech-derived factors, the hirudin-like factors, also called HLFs.

Another important component of the saliva is the lipid distribution. In these are the phospholipases and the lipases, that are present to prevent healing of the wound in the attacked host.

c. Habitat

Medicinal leeches' natural habitat extends from Western, Southern Europe and Ural mountains to the countries close to the Mediterranean. Leeches are amphibious animals, they need to be close to water and land. They live mainly in stagnant water of ponds and marshes, in which they generally swim. On land, they use their suckers⁵.

Fig. 4 shows the perfect habitat for Hirudo Medicinalis is a small pond with mud on the edges. There, they will find and suck the blood of mainly mammals but also amphibians and reptiles. But they also have predators such as insects and larvae. The extensive use of leeches isn’t the only reason to their disappearance at the end of the 19th century. The reduced number of marshland cattle herds as well as pollution and damage of their habitat are also direct factors. H. medicinalis is still present in Turkey, Serbia, Croatia, Ukraine... etc. The highest number of leeches are present on the Black Sea coast. Leeches bred by humans are kept in aquaculture (see “Obtention and Storage”).The major exporters of medicinal leeches are now Russia and Turkey.

^{Fig. 4 - Typical habitat of H. medicinalis}

3 - Medical use by veterinarians

The medicinal leech is used in human medicine for a variety of diseases, mostly forvenous congestion that follow reconstructive surgeries, treatments forosteoarthritis, and other less known therapies. In animals, mostly cats, horses and dogs, the primary indication for leech therapy is the need to salvage tissues whose viability is threatened by venous congestion after surgery, or in case of non efficiency of a traditional treatment. Their salivary secretions contain approximately 100 bioactive substances (of which 30 are known) as well as molecules with anti-edematous, bacteriostatic, and analgesic effects, which is why the use of leeches in veterinary medicine is growing.

a. Obtention & storage

The medicinal leeches used in the treatments of animals must come from special leech breeding farms. It is forbidden to take the leeches from the nature, because they could be contaminated by bacteria, parasites or viruses.

To assure quality on the production site⁶, imported batches must undergo a 32 week quarantine, be traceable by documentation, have had microbial and virological examinations of their water and feeding-blood.

Only healthy leeches are selected; they should be responsive to the touch, motile and flexible. Leeches should be delivered one or two days prior to the treatment, and should look healthy. They are stored in a suitable sterile glass, as shown in Fig. 5, with water that is changed a of couple times per week and they are not fed. Although the glass is lockable, they do not suffocate as their oxygen need is minimal. No more than 5 to 10 leeches should be stored in 1L as their excretion ofmetabolic products could contaminate the batch.

^{Fig. 5 - Storage of a H. medicinalis batch prior to treatment}

b. Procedure

The patients, most commonly cats, dogs or horses, receive one to seven sessions depending on the severity of the disease or wound treated. The number of leeches used in the session varies on the patient specie, its characteristics and its size: one Hirudo medicinalis is used for a small animal weighing 10 kg, whereas therapists use five to fifteen leeches when healing a horse.

According to many reports, it is intuitive for animals with sick hooves to naturally go into leech water. Due to their thermoreceptors, the leeches are led to the hottest areas of the host, which have a high blood supply and/or are inflamed. Some therapists use this behaviour in their procedure. Other therapists place the medicinal leech on the wound, where it pierces the skin with its 3 jaws.

The saliva components of the leech prevent the animal from feeling pain due to the bite. Mucus facilitates lubrication and hydration. The ingestion of blood is helped by the secreted anticoagulant hirudin and sucked by rhythmic contractions of the pharynx. The leeches stay on the affected sites until they reach satiety (5 to 15 ml of blood), which can take from 20-50 minutes in small animals to 120 minutes in horses.

The figure below shows the steps of suction:

^{Fig. 6 - Steps of suction}

When the H. medicinalis finishes sucking blood, it falls off. Digestion of the blood (mainlyhaemolysis andproteolysis) takes several months and is helped by bacterial symbiotes: Pseudomonas hirudins and Aeromonas hyrdrophilia. On the host, bleeding continues from the the site of the bite and it shouldn’t be stopped as it is part of the therapy. The biting-site is bandaged after the blood cesses to flow. To ensure quality of treatments, one leech should not be used repetitively, even on a same patient.

c. Mode of action of the biochemically active substances

Directly after biting the patient, saliva is released into the wound, diluting and fluidifying the host’s blood. Mainly venous blood is sucked, while Hyaluronidase is released to facilitate penetration and diffusion of active substances with various effects into the tissues. It breaks down hyaluronic acid, a major component of the intercellular matrix. Leeches also secrete other important substances that reduce blood viscosity and have anti-cancer properties.

Antialgesic effect:

The leech releases vasodilating and pain-killing substances which haven’t been fully determined yet, prior to the Hirudin secretion. They produce a number of important neurotransmitters such as dopamine, serotonin, acetylcholine and enkephalin, which reduce the perception of pain by the patient and have a relaxation effect on the whole organism.

Anticoagulating effect:

Hirudin is a competitive inhibitor which binds toThrombin, a central factor of the coagulation cascade, taking the place of Fibrinogen, the natural substrate. It also accelerates the release of Factor Xa, that catalyses the conversion of Prothrombin into Thrombin. Factor Xa is dissolved in the plasma where other salivary components can act, such as Antistasin and Ghilanten, which are potent specific inhibitors of the blood coagulation Factor Xa. There are variants of hirudin:

• HV1 comes from the leech’s body and has no anithrombin effect,
• HV2 come from the leech’s head and has a thrombin effect,
• Recombinant hirudin has been produced as an intravenous injectable preparation for injection, useful for patients who cannot tolerate heparin, but is 10 times less active than natural hirudin.

Platelet aggregation is normally mediated by different molecules like ADP, collagen, epinephrine and thrombin. Apyrase hydrolyses the phosphate of ATP and ADP, unabling the aggregation of platelets. Calin and Saratin interfere in the interactions of collagen with platelets and Von Willebrand factor, whileCollagenase separates the collagen chain. This prevents the adhesion between thrombocytes and the vascular walls.

Destabilase is an effective thrombolytic and firbrinolytic agent, by dissolving the ε-(γ glutamyl)-lysine bonds of fibrin. Other molecules inhibit the proteinase enzymes needed for coagulation. For example, both types (A and B ) of Bdellin inhibit chymotrypsin and trypsin. Kalikrein inhibitor alters the actions of kallikrein and therefore the activation of Factor XII, the primary activator of the intrinsic coagulation pathway.

Anti-inflammatory effect:

Bdellin inhibits the protease involved in the spread of inflammation. Eglin is a lysosomal and bacterial proteinase which also exhibits anti-inflammatory effects.

Stimulation of blood circulation:

• The blood flow into the wound area is increased by Carboxypeptidase A.

• Vasdodilatation is caused byacetycholine.

• Proteolytic enzymes of the mast cell of the host are inhibited by LDTI (Leech Derived Tryptase Inhibitor). It is presumed that this prevents injuries of the leech’s sensitive skin on the anterior sucker.

Antibiotic effects:

The viscosity of the interstitial walls is increased by Hyaluronidase, leading to antibiosis.

d. Conditions leading to treatment failure

The transport of leeches must be done correctly. They must not be used if they show any disease indication, such as show any injuries, leave a blood-trail, smell, have a pale skin colour or be covered in slime.

The water in which the leeches are stored must be analysed. Some substances even at low doses, such as Chlorine (often found in tap water), may lead to their death. They are also sensitive to quinine, saccharin and odorous solvents.

Some drugs or ointments used on the patient may also lead the the treatment inefficiency as leeches won’t bite the host. Although leeches can survive in temperature varying from 0 to over 30°C, they can undergo lethal stress in case of thermic choc. This may happen in hot summers and cold winters (when treating an animal outside), loss rate of leeches is quite high.

4 - Conditions that are already treated with leech therapy

“According to the estimates of Bierbertaler Blutegelzucht GmbH⁷, about 20.000 to 25.000 annual leech treatments are performed on animals” The most common indications are hip and elbow dysplasia, acute and chronic arthritis, diseases associated with inflammation of tendons, ligaments, and fascia, diseases of the vertebrae and the treatment of scars.

Some characteristic indications for Hirudotherapy in Veterinary Medicine:

Arthitis: Hirudotherapy has been proved successful when treating arthritis. In early stages, it can help rejuvenate the cartilage degradation, whereas it will help with pain loss and better blood circulation in later stages. Muscles are be stabilised and joints regain a higher loading capacity.

Wound Care: Leech therapy is very efficient⁸, quick and simple on fresh wounds, lowering the inflammations. Pus is either drained quickly or prevented. Old scares can also be treated.

Inflammation: Joint and connective tissue inflammations are treated with a high success rate. The regenerative is long lasting.

Venous congestion: Leeches act by reducing the capillary pressure and increasing the arterial reperfusion of capillary beds. Venous congestion⁹ can happens due to inefficient venous drainage. There is also a condition known as the venous insufficiency¹⁰; difficulties to form an anastomosis between veins and no successful attempt to reattach a venous supply to a flap. If the venous congestion is not fixed quickly, the blood risks to clot, arteries won’t be able to nourish the tissues because of the blood clot and the tissues will die. To prevent this condition, medicinal leeches are applied to the congested flap¹¹. The leech will consume the excess of blood before it falls off. After the leech biting the wound and falling off the wound will continue to bleed for a while due to anticoagulant components of the leech’s saliva. The effect of the medicinal leech is to reduce the swelling in the tissues and to increase the healing status by allowing fresh oxygenated blood to the wound and area around it.

^{Fig. 7 - Treatment of othematoma in a dog with medicinal leeches}

5 - Risks & contraindications

Not all animals can undergo hirudotherapy. A health check should be done before to rule out the following contraindications : absolute hemophilia, anemia, hypotonia, acute infections, immunosuppression diseases like leukemia, fungal skin diseases and pregnancy.

There are generally no side effects, except rarely occurring allergies. Singular cases of cardiovascular disorders, dyspnoea and pale mucous have been reported and can been treated with homeopathis agents.

Only in 10% of cases, do the biting scares stay. The major risk of postoperative use of medicinal leeches is bacterial infections. If the leech had to vomit during the feeding, one if it’s intestinal symbiotAeromonas hydrophilia¹² may infect the host, showing local inflammation ¹³. Many other bacteria have also been identified like Serratia, Vibrio, Pseudomonas genus… Symptoms of infection are lymphangitis, dermo hyperdermitis, severe sepsis, abscesses and gas necrotising myositis. They can occur 24 hours to a month later. Blood-born viruses could also be transferred between patients, but this is limited by not using a same leech between patients.

6 - Benefits

The benefits of Hirudotherapy counterbalance by far the risks. This non invasive treatment has fast and good results, on top of being cheap and generally safe. Most of the time it is the best solution when “conventional” treatments don’t work.

7 - Future projects

Importance of alternative medicine is growing, which plays a major role here. It is impossible to harvest the substances like hirudin for medical use because of their minuscule amounts present in the leeches. Hirudin and other components of the saliva are synthesized using some recombinant techniques. “Mechanical leeches¹⁴” that perform the same functions as the Hirudo medicinalis, have been developed. The mechanical leeches distribute heparin, an anticoagulant and act at a deeper level in the skin. For now, they are not available commercially.

Notes & Figures:

¹: History of hirudotherapy: http://www.amazingleeches.com/history-of-leech-therapy.html http://www.theartofislamichealing.com/leech-therapy-taleeq-part1-history-overview/

²: The Hirudo medicinalis species complex: https://link.springer.com/article/10.1007/s00114-012-0906-4

• New research shows medicinal leeches have been misclassified: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2176162/ Genetic differentiation between H. medicinalis & H. verbana based on random-amplidied polmorphic DNA: https://link.springer.com/article/10.1007/s00436-004-1181-x

³: Protein profiling of leech salivary gland secretion: https://www.researchgate.net/profile/Victor_Zgoda/publication/8401581_Protein_Profiling_of_the_Medicinal_Leech_Salivary_Gland_Secretion_by_Proteomic_Analytical_Methods/links/54c23b710cf2911c7a46cd71/Protein-Profiling-of-the-Medicinal-Leech-Salivary-Gland-Secretion-by-Proteomic-Analytical-Methods.pdf

⁴: Molecules of saliva - Hirudotherapy: Applications & Indications in Surgery - http://www.ejmanager.com/mnstemps/64/64-1327728946.pdf

• Prot & peptide of salivary gland secretion of medicinal leeches H. verbana, H. medicinalis & H. orientalis: https://link.springer.com/article/10.1134%2FS0006297908030127?LI=true

⁵: Video of locomotion: [[https://upload.wikimedia.org/wikipedia/commons/thumb/1/12/20100214_Leech_climbing_door_at_Lake_Leake,_Tasmania.ogv/220px--20100214_Leech_climbing_door_at_Lake_Leake,_Tasmania.ogv.jpg|https://upload.wikimedia.org/wikipedia/commons/thumb/1/12/20100214_Leech_climbing_door_at_Lake_Leake,_Tasmania.ogv/220px--20100214_Leech_climbing_door_at_Lake_Leake,_Tasmania.ogv.jpg|https://upload.wikimedia.org/wikipedia/commons/thumb/1/12/20100214_Leech_climbing_door_at_Lake_Leake,_Tasmania.ogv/220px--20100214_Leech_climbing_door_at_Lake_Leake,_Tasmania.ogv.jpg|https://upload.wikimedia.org/wikipedia/commons/thumb/1/12/20100214_Leech_climbing_door_at_Lake_Leake%2C_Tasmania.ogv/220px--20100214_Leech_climbing_door_at_Lake_Leake%2C_Tasmania.ogv.jpg]]

⁶: Guidline by the German health authority (Bundesintiture fur Arzneimittel & medizinprodukte BfArM, www.bfarm.de)

⁷: www.blutegel.de/egel/export/sites/default/en/downloads_en/pdf/VET_Aurich_Henne_The_Use_of_Leeches_in_Veterinary_Medicine.pdf

⁸: http://www.sciencedirect.com/science/article/pii/S2005290114000028

⁹: https://vcot.schattauer.de/en/contents/archivestandard/issue/1868/manuscript/21092.html

¹⁰http://www.sciencedirect.com/science/article/pii/S0022480408000176

¹¹: LT in flap salvage: systematic review & practical recommendations: https://www.ncbi.nlm.nih.gov/pubmed/27427444:

¹²: More about Aeromonad spp. infection: https://link.springer.com/article/10.1007%2Fs10096-016-2629-5

¹³: http://aem.asm.org/content/74/19/6151.short

¹⁴: More about the mechanical leech: http://www.abc.net.au/science/news/health/HealthRepublish_439856.htm https://web.archive.org/web/20061211152532/http://www.otoweb.org/news_folder/mech_leech/mech_leech_A.htm

* Fig. 1 - Dorsal & ventral view of Hirudo verbana's body (Wikicommons - File:Hirudo verbana2.jpg)

* Fig. 2 - Anterior sucker of the leech (File:Blutegelmeyer.jpg - Wikimedia Commons)

* Fig. 3 - Some morphologic specificities of the leech (File:AmCyc Leech.jpg - Wikicommons)

* Fig. 4 - Typical habitat of H. medicinalis (File:HirudoMedicinalisHabitat.jpg - Wikicommons)

* Fig. 5 - Storage of a H. medicinalis batch prior to treatment (File:Hirudo medicinalis - sanguisuga 1070197.JPG - Wikicommons){{

* Fig. 6 - Steps of suction of leeches (File:Med. Leech suction action. Hirudotherapy. 01.jpg - Wikicommons)

* Fig. 7 - Treatment of othematoma in a dog with medicinal leeches (File:Blutegel-Othaematom.jpg - Wikicommons)

References

Abdullah S., Dar L.M., Rashid A., Tewari A., 2012, Hirudotherapy /Leech therapy: Applications and Indications in Surgery, Archive of clinical experimental surgery, 1(3): p.172-180 http://leeches-medicinalis.com/the-leeches/http://www.ejmanager.com/mnstemps/64/64-1327728946.pdf

Aurich M., Henne A., 2011,The Use of Leeches in Veterinary Medicine, Biebertaler Blutegelzucht GmbH, http://www.blutegel.de/egel/export/sites/default/en/downloads_en/pdf/VET_Aurich_Henne_The_Use_of_Leeches_in_Veterinary_Medicine.pdf

Baskova I.P., Kostrjukova E.S. Vlasova M.A., Kharitonova O.V., Levitskiy S.A., Zavalova L.L., Moshkovskii S.A., Lazarev V.N., 2011, Proteins and peptides of the salivary gland secretion of medicinal leeches Hirudo verbana, H. medicinalis, and H. orientalis, Biochemistry(Moscow), 73(3): p.315–320https://link.springer.com/article/10.1134%2FS0006297908030127?LI=true

Baskova I.P., Zavalova L.L., Basanova A.V., Moshkovskii S.A, Zgoda V.G., 2004, Protein Profiling of the Medicinal Leech Salivary Gland Secretion by Proteomic Analytical Methods, Biochemistry (Moscow) 69(7): p.770-5.

Cooper E.L., Mologne N., 2016, Exploiting leech saliva to treat osteoarthritis: A provocative perspective, Journal of Traditional and Complementary Medicine, https://doi.org/10.1016/j.jtcme.2016.11.005

Kutschera U., 2012, The Hirudo medicinalis species complex, Naturwissenschaften, 99(5): p.433–434

Sobczak N., Kantyka M., 2014, Hirudotherapy in veterinary medicine, Annals of Parasitology 60(2): p.89-92

Verriere B., Sabatier B., Carbonnelle E., Mainardi J., Prognon P., Whitaker I., Lantieri L., Hivelin M., 2016, Medicinal leech therapy and Aeromonas spp. infection European Journal of Clinical Microbiology & Infectious Diseases, 35(6): p.1001-6

Whitake I.S., Rao J., Izadi D., Butler P.E., 2004, Historical Article: Hirudo medicinalis: ancient origins of, and trends in the use of medicinal leeches throughout history, British journal of Oral and maxillofacial surgery, 42(2): p.133-137