Possible detection of physiological state disorders with the help of medicinal leeches Hirudo verbana Carena, 1820.

In the experiment, 160 medicinal leeches of the species Hirudo verbana Carena, 1820 were studied. Medicinal leeches were fed on the blood of animals and people (conditionally healthy and diseased). Four leeches were taken from each animal/person. The animals were studied for 3 weeks. Mortality was mostly observed in the first days after feeding on the blood of the host. We noted mortality, the appearance of constrictions on the leeches' body, the intensity of the host blood spitting from their body. The host's blood was taken from their stomach on the first day after feeding. Hematological and immunological indicators of blood were determined in the taken blood of the host. As a result of the study of the blood of the sick, significant changes were found, compared to conditionally healthy ones. It was manifested by an increase in erythrocytes and leukocytes. The leukocyte formula looked like in most pathological conditions of the inflammatory process. The obtained indicators of the experiment make it possible to quickly assess the presence of physiological disorders in the early stages of the disease.

The effect of blood human, domestic and small laboratory animals on the viability and behavior of the medicinal leech.

Medicinal leeches are used for therapeutic purposes in the prevention and treatment of many diseases. Because they have a large amount of biologically active substances in their body. Each of these substances has many therapeutic effects. In natural conditions, they are mostly are fed of blood by wild animals. In laboratory conditions, the blood of domestic animals is mostly used.  Currently, medicinal leeches are mostly bred in laboratory conditions. Because there are very few of them in nature. They are listed in the Red Book. Scientists of various specialties are looking  for optimal conditions for their life and breeding. That became our research goal. To identify the influence of blood human, domestic animals (pigs and chickens) and small laboratory animals (rats) on the viability and behavior of medicinal leeches Hirudo verbana Carena, 1820 and Hirudo orientalis Utevsky & Trontelj, 2005. According to this, 8 groups of sexually mature animals were formed: 1 and 2 - human blood; 3, 4 - blood of a domestic pig; 5, 6 - blood of domestic chickens; 7, 8 - blood of a non-linear laboratory rat. As a result of the study, it was found that the blood of pigs and chickens is the most suitable for feeding the medical leech for normal life and behavior. Mortality of leeches was observed when feeding on rat and human blood. It should be noted that at the beginning of feeding animals with blood human. The percentage of cannibalism in animals increased.

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.

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.

The influence of the planting time and conditions on the reproductive properties of Hirudo verbana and Hirudo medicinalis.

Factors that affect on the sexual behavior of leeches not only in nature, but also in artificial conditions remain largely unexplored. We did not find data on the behavior of medicinal leeches, which are not placed in time from the aquatic environment in the peat-soil conditions after the appearance of clitella on their bodies. Therefore, the study of this problem has become relevant. For the study, four experimental animals groups were formed: 1 control - medicinal leeches (H. verbana), which were allowed to breed immediately after the appearance of clitella; 2 experimental - medicinal leeches (H. verbana), which were kept in an aqueous medium after the appearance of clitella for a month and then allowed to breed; 3 control - medicinal leeches (H. medicinalis), which were allowed to breed immediately after the appearance of clitella; 4 experimental - medicinal leeches (H. medicinalis), which were kept in an aqueous medium after the appearance of fertilization belts for a month then allowed to breed. The 400 sexually mature healthy medical leeches of two types aged 1.5-2 years were used in experiments. Initially, four individuals were placed into 4-liter containers for sexual intercourse, and after the appearance of differentiated fertilization zones, the control groups were immediately allowed to breed in a peat-soil environment. The experimental groups were left in the same containers for a month and then the survived leeches were allowed to breed. As a result of our research, we found mortality in the experimental groups. From the first week, we found that mortality before breeding of H. verbana was 15.6 ± 2.1% and of H. medicinalis - 14.3 ± 1.5%. Also, 92 ± 0.9% leeches from experimental group died in pet-soil environment sometimes even without cocoon deposition. The experimental group had defective cocoons, but mostly dead offspring.

Indirect effect of substances of the hemophagous parasite Hirudo verbana on the immune system of the host rats.

A positive relationship between the hematophagous parasite-medicinal leech and the host (animals and humans) has been partially shown by many authors. But only direct influence was shown, and how they can influence indirectly has not been studied by anyone. Therefore, it became relevant to analyse the immunological parameters of blood and histological analysis of the central lymphoid organ of female rats on the 30th day after the birth of their offspring, as well as the same indicators in their offspring on the 30th and 60th days of ontogenesis under the indirect influence of saliva substances of the hematophagous parasite Hirudo verbana. The parasite attachments were carried out to male rats before mating; after four attachments, males were added to the females. After pregnancy, the females were removed. The analysis of indicators was carried out according to the goal. We counted the number of erythrocytes and leukocytes, analysed the leukocyte blood count, studied the phagocytic and metabolic activity of neutrophils, histologically analysed the thymus, carried out a statistical analysis. Under the indirect influence of the substances of the parasite saliva, in experimental female rats and their offspring, in comparison with the control group, a significant increase in the number of leukocytes and erythrocytes is observed. The leukocyte blood count in the experimental group remains unchanged in females and their offspring in comparison with the control. When analysing the thymus in the experimental group, the number of lymphocytes significantly increases in females and their offspring. Substances of the hematophagous parasite are able to indirectly positively influence the general condition of pregnant female rats, and later their offspring. It should be noted that the parasite did not directly affect females and their offspring.

The impact of fetal load of Hirudo verbana saline extract antigens on morphometrical, hematological and immunological parameters of rats in the early stages of post-embryonic development

The aim of the study was to determine the immunostimulatory effect of antigens from saline extract of the medicinal leech on the morphometric parameters of the body and major immunogenic organs of rats. Wild-type female rats were administered medicinal leech saline extract antigens intraperitoneally two weeks before and two weeks after mating (in total four injections). The offspring were examined, as were the mothers after feeding the offspring, at 1, 15, 30, 60 days after birth. The morphometric parameters of the animals were recorded. The animals were then decapitated under anesthesia and subjected to the following tests: morphometric parameters of immunogenic organs (spleen and thymus), total white blood cell count, red blood cell count, hemoglobin, color index and leukocyte formula. In addition, the absorption activity of neutrophils was examined: phagocyte index, phagocyte number, phagocytic capacity of the blood, the number of active phagocytes. From day 1, the medicinal leech antigens were associated with increased numbers of white blood cells and red blood cells, elevated hemoglobin level, increased absorption activity of neutrophils; however, the colour index remained in the normal range. These changes indicate that the antigens found in the saliva of medicinal leeches have an immune-stimulating influence on leucopoiesis, erythropoiesis and morphogenesis.