EXPERIMENTAL ECOLOGICAL RESEARCH ON THE RELATIONSHIPS OF PATHOGENIC MICROORGANISMS WITH ALGAE

One of urgent and relevant challenges, facing humanity, is overcoming the consequences of planet climate changes. At present there are evident changes in the seasonal rainfall distribution in Ukraine’s territory, there are more frequent temperature anomalies, experts forecast a rise in the level of the Sea of Azov and the Black Sea, desertifi cation of southern and south-eastern regions of the country. There is also actual threat of considerable decrease in water resources and deterioration of water quality. Due to these conditions, the issues of ensuring the purity of water sources and their safety for economic use become especially relevant.


INTRODUCTION
One of urgent and relevant challenges, facing humanity, is overcoming the consequences of planet climate changes. At present there are evident changes in the seasonal rainfall distribution in Ukraine's territory, there are more frequent temperature anomalies, experts forecast a rise in the level of the Sea of Azov and the Black Sea, desertifi cation of southern and south-eastern regions of the country. There is also actual threat of considerable decrease in water resources and deteriora-tion of water quality. Due to these conditions, the issues of ensuring the purity of water sources and their safety for economic use become especially relevant.
Water is known to be one of the main paths of dissemination for many dangerous infectious and invasive diseases of humans and animals. A special place in this group of pathogenic microorganisms is taken by Leptospira interrogans spirochetes (Stimson 1907) Wenyon (1926). A wide range of hosts [1][2][3][4][5][6] and considerable ecological fl exibility conditions pervasive dissemination of these pathogenic spirochetes. The cases of leptospirosis -a disease, caused by L. interrogans, are EXPERIMENTAL ECOLOGICAL RESEARCH ON THE RELATIONSHIPS currently registered in many countries on all the continents, except for Antarctica [7][8][9][10][11][12][13][14][15][16].
The capability of pathogenic leptospires to exist in fresh water for a long time, forming natural sources of infection, makes these microorganisms extremely dangerous, especially in conditions of defi cient water resources.
At present there are no effective mechanisms of recovering the territory from L. interrogans, except meliorative draining measures. However, such radical actions are not always reasonable both from ecological and economic standpoint. The biological method of combating pathogenic microorganisms in environmental objects opens new perspectives in this direction. However, its elaboration and application require the clarifi cation of many issues, related to the existence of L. interrogans in different types of freshwater sources and the place of these microorganisms in a complicated network of biotic relationships between hydrobionts.
A considerable part of primary biological products in freshwater bodies is created by different species of algae. They are also one of the main sources of biologically active substances (BAS) for hydrobiocenoses, ensuring complicated allelopathic interactions between higher plants, algae and bacterial microfl ora.
Water bodies are an extremely complicated and dynamic environment for L. interrogans [15] which creates considerable methodological diffi culties in planning, conducting experiments, and interpreting the obtained results in situ. The aforementioned and insuffi cient scientifi c data create conditions, due to which the study of ecological relationships between pathogenic microorganisms (L. interrogans) and freshwater algae should be conducted in in vitro experiments under controlled laboratory conditions.
The main aim of this study was to investigate the specifi cities of ecological relationships between patho-genic leptospires and algae -Chlamydomonas proteus Pringsheim 1930, and to compare the response of different serological types of L. interrogans to the effect of BAS, produced by this species of algae. Hopefully, the obtained data will facilitate more complete investigation on the specifi cities of biotic relationships of pathogenic leptospires in natural water bodies and provide suffi cient data for further elaboration of effi cient methods to decrease the potential of natural leptospirosis sources.

MATERIALS AND METHODS
Unialgal monocultures of green algae, С. proteus, were cultivated in Erlenmeyer fl asks of 250 cc on the Fitzgerald's medium in the Zehnder and Gorham's modifi cation [18] at 22-25 ºС and 12-hour-long photoperiod of artifi cial illumination with 25 klx fl uorescent lamps.
The Terskih and Korthof's medium, containing 10 % inactivated sheep blood serum, was used to cultivate leptospires.
The cultures of spirochetes of 7-14 days with the accumulation of 50-100 leptospires per vision fi eld, with characteristic morphology, active mobility and no signs of autoagglutination were used in the experiment. The experiments were conducted with cultures of the following strains of leptospires (Table 1), which are most widespread in Ukraine's territory, and used as antigens during laboratory diagnostics of leptospirosis in the serological reaction of microagglutination and lysis.
Culture solutions of algae were passed through sterile cellulose fi lters with pore diameter of 0.2 μm (Sartorius, Germany). This method of sample preparation allowed removing the symbiotic microfl ora, notable for C. proteus cultures, and preventing the destruction of biologically active substances (BAS), released by algae.
The experiment, studying the allelopathic effect of green algae on pathogenic leptospires, simulated the conditions of freshwater bodies on the territory of leptospirosis sources. In particular, the gradient of BAS concentration, released by algae in natural conditions, was presented in experimental samples by the following dilutions of C. proteus fi ltrates -1 : 10, 1 : 100, 1 : 1,000, and 1 : 10,000. Control samples contained sterile culture Fitzgerald's medium in the Zehnder and Gorham's modifi cation.
The samples were introduced the same volume of pathogenic leptospire cultures, here the inoculates of each serological type were taken from one volume. It ensured the same density of leptospires in the experiment and control at the beginning of the experiment. The study was conducted in fi ve repeats. After 24 h since the beginning of the experiment, the content of spirochetes was determined in the experimental and control samples, using direct calculation in 40 μm chambers.
The character and explicitness of the effect of green algae, C. proteus, secretions on pathogenic leptospires was evaluated, comparing the content of spirochetes in the experimental and control samples, here the density of cultures in the control was accepted as 100 % [19].

RESEARCH RESULTS
Explicit inhibition of experimental cultures of L. interrogans was observed in the experimental samples, containing cultural fi ltrates of С. proteus in 1:10 dilution (Table 2). For instance, after 24 h since the be-ginning of the experiment the content of leptospires in the experimental samples was as follows (% from the control): Tarassovi - Using the criteria of estimating the effect of ecological factors on populations (cultures) of microorganisms [19], we would like to note that according to the experiment results, the leptospire cultures of serological types Pomona, Canicola, Hebdomadis, Sejroe, Icterohaemorrhagiae, Grippotyphosa, Australis were exposed to moderate inhibition due to the allelopathic effect of С. proteus. The leptospire cultures of serological type Tarassovi had weak inhibition. According to the increasing sensitivity to the allelopathic effect of C. proteus, serological types of leptospires formed the following row: Tarassovi  Lower density of L. interrogans, compared to the control, was noted in another group of experimental samples, containing cultural fi ltrates of С. proteus in 1 : 100 dilution. For instance, it was determined that the density of leptospire cells in the tests, taken from the experimental samples, was as follows (% from the control): Tarassovi     In the subsequent experiments with an even higher dilution index for cultural fi ltrates of C. proteus -1 : 1,000, the difference between the density of leptospire cultures in the experiment and control was as follows: Tarassovi Table 4).
The results demonstrated that according to the accepted criterion of estimating the effect of ecological The data, obtained during the experiments of studying the effect of C. proteus fi ltrates in 1 : 10,000 dilution on the cultures of serological groups of leptospires under investigation, demonstrated that there was no statistically reliable difference between the density of L. interrogans cells in the experiment and the control (Table 5). Therefore, at this concentration of BAS, secreted by green algae, in the aqueous medium, leptospires were not affected by the allelopathic impact from them.

DISCUSSION
The results of studies demonstrated that the pathogenic spirochetes of L. interrogans responded explicitly to the allelopathic effect of green algae С. proteus only under suffi ciently high content of BAS, secreted by the latter during their existence, in the aqueous medium (1 : 10 -1 : 100). Similar conditions may occur in natural sources of leptospirosis, water bodies, during a warm season in the period of mass propagation of this type of algae, when the content of BAS, secreted by them, is the highest. As green algae С. proteus affect L. interrogans spirochetes via the change in characteristics of their existence medium, ecological interspecies relationships between them should be deemed topical. At the same type, it should be noted that leptospire cultures of serological types, used in the studies, demonstrated different sensitivity to the presence of similar concentrations of BAS from algae in the medium. Thus, according to the increasing sensitivity to the allelopathic effect of algae, the investigated serological types formed the following row: Tarassovi, Icterohaemorrhagiae, Pomona, Grippotyphosa, Australis, Sejroe, Canicola, Hebdomadis. The mechanisms, conditioning similar differences in the response to ecological factors of different serological groups of leptospires, are yet to be studied in the full detail. However, their adaptive signifi cance was absolutely evident -a complicated intraspecies structure of L. interrogans determined wide ecological fl exibility of this species, which allows it to exist in different environmental conditions and ensures the resistance to the effect of many ecological factors.

CONCLUSIONS
In the experiment, a topical type of ecological interspecies relationships is formed between L. interrogans and green species of C. proteus algae, which is realized through the release of biologically active substances into the habitat by C. proteus. Explicit inhibition of leptospire cultures under investigation was observed only in the samples with low dilutions of 1 : 10 -1 : 100 algae fi ltrates. Pathogenic leptospires practically did not respond to the presence of BAS from algae in the medium under the fi ltrate dilution indices of 1 : 1,000 -1 : 10,000. The serological types of leptospires, used in the experiment, demonstrated different sensitivity to the presence of BAS, secreted by C. proteus,