In vitro cultivation of Leishmania donovani promastigotes: Growth potential of human urine as replacement of fetal calf serum

The in vitro cultivation of protozoan parasites involves highly complex procedures, which are subject to many variables. Some of these parasites have a very complex life cycle that may require different culture parameters [1]. The culture medium is used to simulate the biochemical environment found within the sand fl y vector [2] and there are different researches to make the optimal ones that used in various purposes e.g. diagnosis, immunologic and molecular testing and vaccination [1]. A variety of culture media have been used to cultivate Leishmania; these can be divided into three main categories: semi-solid, biphasic media and liquid monophasic media [3]. Since biphasic and semisolid culture media need blood, an important factor for the reproduction of parasites, which was later enriched with bacteriologic additives such as brain heart infusion is used with various modifi cations of the liquid phase added to the solid phase [4]. Most liquid media may require Fetal Calf Serum (FCS) or erythrocyte lysate, but some recent studies suggested serum-free liquid medium [3,5]. The Isolation of Leishmania promastigotes from culture is not always successful and this may be due to several factors related to the interaction of parasites with the ingredients that supplemented to media. These stimulate the attempts Abstract

Citation: Sharief  of modifying the culture media by various components to enhance the growth of promastigotes that lead to increasing of their numbers [6]. As promastigotes growth need nutrients, stimulatory components, and good environmental conditions i.e. sugar is considered as a source of carbon, proteins as source of amino acid, and lipids as source of energy [7][8][9].
Moreover other stimulatory components governed by suitable temperature are vital for good growth [10,11].
Several studies have shown the stimulatory effect of human urine on Leishmania promastigotes [11][12][13]. When supplemented with human urine, Schneider's Drosophila culture media was found to increase the prolif eration of different Leishmania parasite species. In the same study, it was found that culturing amastigotes that were isolated from Leishmania -infected hamsters in a culture environment containing urine increased promastigote differentiation and proliferation compared with controls [11]. Another study questioned whether urine from a healthy human donor, a patient with nephritis, and several types of animals could be an alternative to Fetal Calf Serum (FCS) for in vitro L. donovani culture. Results have indicated that urine was not a valid alternative to FCS, but can be used to increase the proliferation in L. donovani cultures [12]. to cool to 55 °C before 10% defi brinated rabbit blood was aseptically added gently mixed with the contents by rolling, then 1% of penicillin (50 μg/ml), and streptomycin (50 μg/ml) solution was added to the blood agar mixture. Slopes of culture medium were then prepared by dispensing 2-3 ml of the blood agar mixture into sterile containers that were then set in a slope position until the agar completely solidifi ed. Full fatty and skimmed cow milk were purchased from the local market.

Materials and methods
Three grams from each milk form was added separately to 30 ml of distilled H 2 O and sterilized by autoclaving at 110°C for 10 minutes. Agar was added to milk at the same concentration similar to that used in preparing blood-agar medium and other procedures were followed. Five liquid overlays were freshly prepared before being added to the solid phases of the culture media. These were 5% dextrose, 10% full fatty milk, 10% skim (non fatty) milk, 5% human urine and normal saline.
Both NNN and milk agar media were supplemented with 20% heat-inactivated Fetal Bovine Serum (FBS) for control cultures.
Urine samples were collected from 10 healthy apparently healthy men who had normal prostate, kidney and bladder, and had tested negative for HIV and urinary schistosmiasis on several occasions. Urine samples were taken in the morning before any food or drink was consumed. All urine samples were sterilized by 0.22μm fi ltration and stored in sterile falcon tubes at 4°C. An aliquot of 1x10 7 parasites/ml was inoculated into each culture vessels containing solid medium overlaid with 2 ml of each liquid phase and incubated at 26 o C. Cultures were daily seen and the growth and behavior of parasites in the media will be observed daily and the media will be change after 3 days. After day 6 of cultivation the overlays were centrifuged to pellet the parasites, supernatants were discarded and each solid medium was overlaid with free liquid phase. Leishmania count was adjusted to 1x10 6 parasites/ml and inoculated into each culture container. All cultures were incubated for 12 days at 26°C and the growth was counted at days 4, 8 and 12. The parasites was counted by adding 100 μl from each overlay to 1 ml of 5% formalin (25 ml of 0.9 % NaCl + 4.1 ml fromaldyhide), using counting chamber.

Results
Cell cycle parameters and proliferation index of L. donovani in medium supplemented with 5% urine are shown in

Discussion
Inoculation of Leishmania in NNN medium was considered as our measuring scale for parasite growth in modifi ed media.
Although addition of blood may render blood-agar media to become more susceptible to contamination but it is still be as an essential component of vital importance to parasite growth.
In this study, cultivation of Leishmania parasites in NNN medium was considered Addition of female human urine to this medium enhanced the growth of the promastigotes by at least 40 times when compared to the classical NNN medium. Such signifi cant increase in parasite growth has previously been reported by several authors who substituted serum with mammalian urine for enhancement of various Leishmania species [13][14][15][16]. Furthermore, Warburg, et al. [12] found that 5% of human urine has contained 10μl xanthine that had signifi cantly enhanced the growth of Leishmania major promastigotes in vitro. However, increasing the concentration of urine up to 4% exhibited no satisfactory effect on the growth of the parasites. This later observation seems to contradict with our results in which the concentration of urine was 5% and in spite of that it did not retard the growth of the parasites.  promastigotes was statistically declined which refl ects an inhibitory and/or lethal effect.
This may also indicates an enrichment and metabolic enhancement factor that might be contained in the fatty milk. Furthermore, other milk components were also studied in regards to their infl uence in the parasites in vitro growth.
Tyndalized milk of cow, goat and buffalo were found to possess some components that may replace fetal bovine serum during the cultivation of Leishmania donovani promastigotes [8].
Another observation in our study was that urine concentrations greater than 5% may inhibit proliferation of Leishmania parasites [16][17][18]. In our study, distinct from the other studies, the reason for increased development of parasites in culture containing 5% urine may be dependent on use of parasite species and types of culture medium.