International Journal of Medical Laboratory

Leishmaniasis is an ancient disease and indications as to craving facial infections can be found on ceramic containers excavated in Central America dating as far back as 1,000 years and on pictures of the Spanish conquerors in the 16th century [1]. Cutaneous leishmaniasis has been known since ancient times and it is truly a neglected disease. Bray referred to a tablet in the library of King Ashurbanipal of Assyria in Neinava (Mosul, Iraq), with the description of a painless ulcer most likely cutaneous leishmaniasis, translated from a tablet of the old Akkadian period in the second or third millennium BC [2].
The cell culture technique is an approach to make efforts to prepare complex habitat conditions of living organisms to developing our knowledge about their behavior and finding suitable ways (like effective vaccine) in order to prevent their negative side effects. On the other hand, routine commercial culture media for cultivation of Leishmania such as Roswell Park Memorial Institute (RPMI)-1640, medium 199, and Schneider's Drosophila, enriched by fetal calf serum (FCS) or blood lysate [3, 4], are many expensive. FCS is highly expensive, and its reliable supply is very difficult to obtain especially in the developing countries [5].
Many attempts have been made to replace FCS in Leishmania culture media with different kinds of sera, bovine serum albumin, a mixture of purine bases, vitamins, large concentrations of certain amino acids, hormones, hemin, hemoglobulin, human and animal urine and, more recently chicken serum [6-15] the later of which introduced an alternative low-cost serum that can be used in culture media for primary isolation, routine cultivation and mass cultivation of Leishmania parasites [6, 7].
Some investigations indicate that the Iberian hare (Lepus granatensis) has been described as a new competent reservoir of the visceral leishmaniasis and its ability to transmit the parasite to sandflies was demonstrated by xenodiagnostic studies which confirmed that Iberian hares can contribute to the outbreak of disease [16-18]. A study found theprevalence of L.infantum infection to be 43.6% from principally two hare species (L.granatensis and L.europaeus) [19] and in another a seroprevalence of 46% was also reported in rabbits [16].
In regard with the above result on the susceptibility of rabbit to leishmaniasis and potential of suitability of rabbit serum for cultivation of Leishmania parasites, and because rabbit blood is an essential component of conventional NNN medium [20], we describe a formulation for cultivation of promastigotes of Leishmania major.
Materials and Methods
Media preparation
For the Luria-Bertani (LB) broth, 1 g peptone, 0.5 g yeast extract and 1 g NaCl were suspended in 100 ml of distilled water and complete medium autoclaved for 15 min at 121°C and were left to cool down and then 5 ml of it was added to lyophilized rabbit anti-sheep cell haemolysin vial (Razi Vaccine and Serum Research Institute, Islamic Republic of Iran) according to the manufacturer's protocol for reconstitution of it and were all mixed thoroughly. After reconstitution of lyophilized rabbit serum, dilutions of it in LB broth were prepared as follows: 1%, 2.5%, 5% and 10%. RPMI-1640 supplemented with heat-inactivated (56°C for 30 min) fetal calf serum (10%) was used as a standard medium for proliferation pattern comparison. No antibiotic was added to the media; they were kept at 4°C.
Continuous cultivation of the promastigotes
The procedures were performed according to the our previous experiences [21]. The Mid-log phase promastigotes of Leishmania major (MRHO/ IR/76/ER) which had been previously grown in RPMI-1640 supplemented with 10% FCS were concentrated by centrifugation at 2500 g for 10 min and washed twice with sterile phosphate-buffered saline solution (PBS) to remove any FCS traces. Parasites were counted using a Neubauer chamber (Haemocytometer) slide and diluted in PBS to a final concentration of 10⁸ parasites per milliliter. Subcultures from each dilution of rabbit serum were performed in 3 repeated series and in each one, triplicate cultures prepared, alongside of the positive control (RPMI-10% FCS), and all the experimented media were inoculated with mid-log phase promastigotes at the final concentration of 5×10⁶ promastigotes/ml in 25 cm² plastic culture flasks. Every flask totally contained 10 ml of parasites and complete media mixture. The flasks were placed in an incubator at 26˚C. The continuous cultures of the parasites were made weekly by sub-culturing into fresh medium until they reached the 10^th sub-culture. In all the sub-cultures, parasites proliferation was assessed both qualitatively and quantitatively by microscopic observations and Giemsa slide preparation. The number of parasites was counted daily by using the Neubauer chamber slide.
In vivo infectivity determination of cultivated parasites
Stationary-phase promastigotes of Leishmania major (2×10⁶ parasites) from the 4^th subculture in new formulation medium were washed twice in sterile PBS and were inoculated subcutaneously into the tail base of 10 female BALB/c mice (6-7 weeks) with 10 mice serving as negative (inoculated with PBS) and positive (inoculated with parasites grown in RPMI-10% FCS) control. The animals were evaluated macroscopically every day until the appearance of lesions.
This research was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Razi Vaccine and Serum Research Institute. All animals experiments, including maintenance, handling and blood collection were approved by Institutional Animal Care and Research Advisory Committee of Razi Vaccine and Serum Research Institute based on the Specific National Ethical Guidelines for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education of Iran.
Statistical analysis
SPSS version 22 for windows® was used for data evaluation. The differences between the averages of the quantitative variables were evaluated by Student’s T-test and the value of p<0.05 was accepted as statistically significant.
Results
Assessment of the parasite growth quantity and replication pattern
It was observed that the addition of 1-10% lyophilized rabbit serum to the LB broth medium significantly stimulated the growth of the promastigotes of Leishmania major their effects of which were comparable to the growth pattern of the parasites grown in the RPMI-1640 -10% FCS (p<0.05). Under invert microscopy, it was also observed that the promastigotes were elongate and had rapid motility. Fine grown parasites and typical morphology of the promastigotes were observed in Giemsa-stained smears prepared from culture media of all serum dilutions. Parasites inoculated into the culture media took about 7 days to reach to the late log-phase. It was also observed that the new formulation supported the continuity of the parasites in successive passages. The effect of various concentrations of the lyophilized rabbit anti-sheep cell haemolysin in the culture media on the growth quantity of the promastigotes and a typical growth curve of the parasite growth in all dilutions of lyophilized rabbit serum is shown in figure 1.
 
 
 
In vivo assessment of pathogenicity of cultivated parasites
Four weeks after infection of BALB/c mice, typical cutaneous lesions of leishmaniasis were observed in the tail base of all experimentally infected animals. Samples of the lesions were taken and evaluated by Giemsa stain for parasitological confirmation and the presence of amastigotes was confirmed in all lesions.
Discussion
The leishmaniases are a group of human diseases caused by kinetoplastid protozoa of the genus Leishmania. Despite having a long history, dating back from more than 4500 years according to ancient descriptions [22], leishmaniasis still ranks at the top three of the neglected tropical diseases caused by protozoa [23]. There are about 1.5-2 million new cases and 70 000 death reports each year, and 350 million people are at risk of infection and disease with about 2.4 million disability-adjusted life-years [24].
The cultivation of Leishmania and other hemoflagellates has been a subject of much interest due to the necessity of performing biochemical and immunological studies with isolated parasites in an effort to develop future therapeutic and preventive tools [25]. In vitro cultivation of these parasites have progressed from crude media with undefined ingredients to fully defined media with serum substitutes capable of supporting good to excellent growth of the organisms [11]. Generally, the media used for Leishmania cultivation can be divided into two categories: biphasic and monophasic. Handling biphasic media are substantially more technically demanding than handling liquid media, which are more suitable for obtention of large amounts of the parasites [26]. Recently, Nasiri had a new insight out of an old NNN biphasic medium to cultivate Leishmania on sheep blood-LB agar base medium (SLM) as a simple and suitable medium for obtaining Leishmania major promastigotes [27].
Several studies have investigated the presence of Leishmania in livestock animals [28-33]. Herman studied the effect of rabbit, chicken, man, calf, hamster and cotton rat sera on the number and morphosis of L.donovani as well as cells in cultures of hamster-peritoneal macrophages that had been infected In vivo [9]. A research on finding a replacement for FCS in cultivation of promastigotes of Leishmania major and Leishmania infantum indicated that chicken serum is suitable for the nutritional requirement of parasites and indicates a potentiallty new medium to be used in long-term In vitro cultivation of Leishmanian promastigotes [6, 7]. In previous studies it was found that the serum and the urine of many animals like hamster, rabbit and sheep are suitable for cultivation of promastigotes. However, collecting of the serum of some of these, like hamster is expensive and using of some of them, like sheep serum, is accompanied with many challenges about adaptation problems of promastigotes to new serum [34].
The LB broth-lyophilized rabbit anti-sheep cell haemolysin medium has some advantages, including the readily availability of chemical compounds, simple storage conditions and low cost preparation. LB broth and its ingredients are readily available in every laboratory at low-cost and its preparation does not require sophisticated, expensive equipment like filtering system and can be prepared by autoclavation [21]. It has been shown that unlike the cow, horse and dog serum that have negative effects on growth behavior of promastigotes, lyophilized rabbit anti-sheep cell haemolysin, like its blood, is suitable for the nutritional requirement of parasites. According to the manufactures instructor, we could storage lyophilized rabbit anti-sheep cell haemolysin at 4˚C prior to restoration and this is one of the main advantages of this medium.
Conclusion
This study has introduced a potent low-cost medium that can be used in cultivation of promastigotes of Leishmania major.
Conflict of Interest
We declare that we have no conflict of interest.
Acknowledgment
The authors are thankful to the director of RVSRI for providing the facilities for the research.

 
 
References