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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2020 Feb 8;44(2):299–304. doi: 10.1007/s12639-020-01203-x

Comparison of camel, dog and the laboratory animals’ sera with the fetal calf serum (FCS) for cultivation of Leishmania major

Frideh Esfandiari 1, Amin Derakhshanfar 2,3, Fatemeh Goudarzi 1, Gholamreza Hatam 4,✉
PMCID: PMC7244685  PMID: 32508404

Abstract

The best-known serum for Leishmania spp. cultivation is the fetal calf serum (FCS), which is very expensive with ethical concerns. This study was conducted to compare various laboratory (BALB/c mice, rat, rabbit, hamster and guinea pig) and non-laboratory (dog and camel) animals’ sera as a substitute for FCS in L. major culture. L. major, MRHO/IR/75/ER strain, was cultivated in RPMI-1640 medium enriched with different percentages of mentioned animal’s sera. Parasite growth was checked constantly. The rate of growth and survival of parasites were compared with a control medium enriched with FCS. As well, biochemical (albumin, globulin AST, ALT, ALP, Bil, BUN, Crea, Ca, P, Na, K, Fe, TIBC, Mg, zinc, Chol, HDL, LDL, TG, BS, uric acid, LDH, CPK) analysis of all sera was performed and compared with FCS. The most promastigote growth rate is considered in 10% BALB/c, guinea pig and hamster sera on the 6th day of cultivation. Also, on the 8th day, parasites showed viability in all animal sera. The promastigote growth in culture media enriched with the camel and the dog sera in comparison with laboratory animals was considered very low. Differences between 10% FCS and 10% cocktail serum were not significant (p > 0.05) but with other sera were significant (p < 0.05). Also, differences between BALB/c with hamster and guinea pig sera were not significant, respectively (p = 0.07 and p = 0.09). According to the biochemical analysis of all sera, the higher content of iron was detected in the hamster, guinea pig, BALB/c and fetal calf sera. The magnesium and zinc content of guinea pig and BALB/c serum was found to be more than the others and comparable with FCS. The promastigote growth decreased by camel, dog and rat sera orderly. In this study, a rapid increase in parasite growth in media supplemented with hamster, BALB/c and guinea pig sera was considered. It could be suggested to use these sera as a suitable alternative for FCS in molecular biology researches.

Keywords: Leishmania major, Laboratory animals, Fetal calf serum

Introduction

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus, Leishmania, which is the third most important vector-borne among tropical diseases with 12 million annual incidences (Limoncu et al. 2004; Esfandiari et al. 2018). Protozoan parasite of the genus Leishmania transmitted to human (over 20 Leishmania species) via the bite of the infected sandfly, causing leishmaniasis. The infectious stage of parasite exists extracellularly in the female sandfly insect vector gut and culture media, and the aflagellated form of parasite resides intracellularly within the vertebrate host cell. Cutaneous, mucocutaneous, visceral and diffuse cutaneous are four main clinical forms of leishmaniasis (WHO 2017). According to the WHO records, the disease is epidemic in at least 88 countries, that of which 72 are developing. An estimated 700,000–1 million new cases and 20,000 to 30,000 deaths occur annually (WHO 2017, 1991; Bessat and Shanat 2013). Leishmania culture media categorized into monophasic (liquid) and biphasic media (Grekov et al. 2011). In research projects and diagnostic purposes of Leishmania spp., various methods such as the enzyme-linked immunosorbent assay (ELISA), isoenzyme characterization, proteomics analysis and the indirect immunofluorescence antibody test (IFAT) are depend on the cultivation of microorganism, especially in liquid media, which is one of the most important requirements of the work (Hatam et al. 2009; Rashidi et al. 2018; Bahrami et al. 2011). Nowadays, fetal calf serum (FCS) is the most common supplement used as liquid media for mass cultivation, because of its high content of growth-promoting factors. FCS is produced by commercial companies at a very large scale, so that the FCS is produced as much as 500,000 L per year, which is obtained from 1,000,000 fetal bovine (Gstraunthaler 2003; van der Valk et al. 2004) and is a wealthy source of often components such as micronutrients, attachment factors, water-insoluble nutrients, trace elements, growth factors, hormones, proteases and protective elements. There are several factors that indicate the need for a suitable alternative for FCS. These include batch-to-batch variation in composition which leads to inappropriate growth and productivity, possibility of viruses, mycoplasma, prion and antibodies contamination, toxicity of the serum at high concentrations, high price and ethical concern (Nasiri et al. 2011; Torres and Ortega 2006). Hence, during the last years, due to restrictions on the production of FCS, many studies have been done to find a suitable alternative to FCS in cultures. These include hydatid cyst fluid, cattle intraocular fluid, cow and buffalo milk, cattle colostrum, egg yolk, protein fractions from plants, the serum of human, horse, pig, chicken, sheep, goat, cow, rat, mouse and other blood derivatives (plasma, platelet lysate and albumin) and human urine, which have been somewhat successful (Gstraunthaler 2003; Nasiri et al. 2011; Fakhar et al. 2012; Sasse et al. 2000; Schwartz et al. 1984; Filipic et al. 2002; Muniaraj et al. 2007; Ferreira et al. 2007; Goodarzi et al. 2014). Using laboratory animals is preferable because of their availability and determination of exact age in comparison with the non-laboratory (camel and dog sera) animals. Therefore, we aimed to find a suitable substitution for FCS in L. major cultivation.

Materials and methods

Parasite cultivation

WHO reference strain L. major (MRHO/IR/75/ER) was used for in vitro study. Firstly, L. major was maintained by a continuous passage through inbred BALB/c mice (6–8 weeks old) that were obtained from Pasteur Institute of Iran. Then, the lesion biopsy was transferred to biphasic medium Novy–MacNeal–Nicolle (NNN) for isolation of promastigotes. Secondly, they were subcultured in RPMI-1640 medium (Sigma, USA) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in a 25 °C incubator for mass cultivation.

Serum collection

In this study, animals would be housed under identical conditions. Blood samples of laboratory animals, including BALB/c, rabbit, hamster, guinea pig and rat, were obtained from the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Also, dog and camel blood samples were collected from the School of Veterinary Shiraz University, Shiraz, Iran. The sera was centrifuged at 1210g for 10 min and inactivated at 56 °C for 30 min. As well, a part of sera in comparison with FCS without filtering was set aside in order to perform biochemical analysis tests. The rest of the sera was filtered with a 0.22-μm Millipore filter and stored at – 20 °C until use.

Cultivation in supplemented media

Nine sets of RPMI-1640 medium with 100 U/ml penicillin and 100 mg/ml streptomycin and growth supplements were prepared as follows: set A, the classic culture media (10% FCS), set B (10% BALB/c serum), set C (10% rat serum), set D (10% guinea pig serum), set E (10% hamster serum), set F (10% rabbit serum), set G (10% dog serum), set H (10% camel serum) and set J (cocktail serum from hamster, guinea pig and BALB/c). 1 × 106 promastigotes of L. major in the logarithmic phase were subcultured in nine set media separately and placed at 25 °C in an incubator. The parasites were counted per 12 h for a week with the Neubauer chamber. In similar conditions, all these experiments were performed three times with three passages of cultivation. In the next step of the experiment, a comparative study of growth stimulation was performed to examine whether all sets from various sera have various stimulating effects. All other biochemical assays were carried out with an Ektachem 400 clinical analyzer (Eastman Kodak Co., Rochester, NY).

Experimental evaluation

Ten BALB/c mice (4–6 weeks old) were purchased from Pasteur Institute (Iran). Promastigotes were harvested at the stationary phase from a medium supplemented with 10% FCS as control and 10% cocktail serum. Four weeks after intradermal inoculation of the parasite to BALB/c mice, cutaneous lesions appeared on the site of inoculation (base of the mice tail). The lesion biopsy was prepared and transferred to biphasic media for in vitro isolation of the parasites, and after well growing, one million promastigotes of L. major in logarithmic phase were subcultured in the RPMI media supplemented with 10% FCS and 10% cocktail sera separately and incubated at 25 °C. The parasites were counted per 12 h for 8 days with the Neubauer chamber. In similar conditions, all these experiments were performed three times with three passages of cultivation. In the next step of the experiment, a comparative study of growth stimulation was performed.

Statistical analysis

Prism 6.0 (GraphPad Software San Diego, Calif., USA) was used to evaluate the data. Student’s t test was appraised to compare promastigote growth results between two experimental groups. Multiple comparisons between more than two groups were made by analysis of variance (ANOVA). A p value of < 0.05 was considered to be significant.

Results

We compared the growth of L. major in different supplement culture media, and repeated measures one-way ANOVA test was performed for analysis and is shown in Fig. 1. Also, the differences between both the 10% FCS and 10% cocktail serum are indicated in Fig. 2. The highest growth rate of the parasite in each passage was observed on the 6th day in sera of 10% BALB/c, hamster and guinea pig, and the lowest survival rate of the parasite on the 8th day in dog and camel serum (Fig. 1). Differences between 10% FCS and 10% cocktail serum during 8 days were not significant (p > 0.05) except the 4th and 5th days (Fig. 2), but with other sera were significant (p < 0.05) in every 8 days. Also, differences between BALB/c mice with hamster and guinea pig sera were not significant, respectively (p = 0.07 and p = 0.09). On the other hand, there is a significant difference between 10% cocktail serum with 10% dog and 10% camel serum, respectively (p value = 0.03 and p = 0.04). Furthermore, differences were significant (p = 0.01 and p = 0.04) between 10% FCS with 10% dog and 10% camel sera, respectively. Also, according to in vivo assessment, this cocktail serum was suitable for parasites isolation from infected BALB/c mice and its comparison with 10% FCS was not significant (p = 0.06). Also, based on biochemical findings, the amount of iron in the serum of hamster, guinea pigs and BALB/c was higher than the rest. Magnesium and zinc levels were higher than in guinea pig and BALB/c, and cholesterol level was higher in BALB/c than in other sera.

Fig. 1.

Fig. 1

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Comparison of L. major promastigote growth in nine set media. The classic culture media of parasites (RPMI-1640 with 10% FCS) are compared with other set media

Fig. 2.

Fig. 2

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Comparison of L. major promastigote growth in the classic culture media of parasites (RPMI-1640 with 10% FCS) and 10% cocktail serum (guinea pig, BALB/c and hamster)

Discussion

Animal sera have been extensively investigated in various in vitro studies. Researchers from 30 years ago started efforts to find substitution for animal serum (Hayashi and Sato 1976). Since the harvesting and production of FCS are ethically problematic (Van der valk et al. 2004; Brunner et al. 2010), a replacement is needed. Supplying all necessary nutrients is important for growth, proliferation, attachment, expansion and maintenance of cells. Serum, in comparison with plasma, more promotes the growth and proliferation of cultured cells (Gospodarowicz and Charles 1980). The major serum components in culture media are hormones, essential minerals, trace elements, growth factors, lipids and proteins (Hill and Treisman 1995). The basic medium for cell culture should be supplemented with specific factors, like hormones, growth factors, fatty acids, lipids, vitamins and trace elements (Taub 1990). In this study, a rapid increase in parasite growth in media supplemented with hamster, BALB/c and guinea pig was considered during the first week and the number of promastigotes in the media reached the same level of the control medium (supplemented with 10% FCS) on the same day, but that in the media supplemented with dog, rat and camel serum was reported as low. The differences between 10% cocktail serum media with 10% rat, 10% rabbit, 10% dog and 10% camel were not found to be statistically significant. This study demonstrated some outcomes with the replacement of FCS with animal sera. For example, the availability of the laboratory animals could help the researchers to prepare easily the appropriate supplements for the culture medium, especially for isolation and molecular detection of the Leishmania parasite that does not require mass cultivation. This study showed that the use of serum of camel, rat and dog is not suitable as a supplement for the treatment of Leishmania parasites; perhaps, due to more exposure to external factors, laboratory animals were kept away in a standard environment. Detoxification is required to eliminate the endotoxin of bacteria, a process that is considered in the preparation of FCS. Also, research in this field has shown that cholesterol, iron, magnesium and zinc play a vital role in the function of Leishmania (Pucadyil et al. 2004). Since the protozoa are not able to synthesize cholesterol, it should be added to the culture medium as a supplement. Lipids are important in the maintenance of many cellular structures and the survival of intracellular parasites. Our research has shown that the serum of mice contains more nutrients for the growth of the parasite. Based on biochemical findings in the study, the amount of iron, magnesium, zinc and cholesterol in hamster, BALB/c and guinea pig sera was higher than in other sera. On the other hand, the role of zinc and magnesium has been proven in cell growth and regulation of blood glucose levels. Therefore, the use of laboratory animals’ sera is appropriate for isolation and is not suitable for mass cultivation. It will also be used for the study about the parasite biology and molecular investigations that will require little of the samples. The effects of many animals’ sera, including rat, hamster, rabbit, chicken and calf, have been studied on the L. donovani by Herman (1966). According to the results obtained in this study, the level of these substances can be higher in the serum. Nasiri et al. showed that the growth of promastigotes of L. major with RPMI-1640 enriched with 10% chicken serum was approximately equal to RPMI-1640 enriched with 10% FCS. Some important limitation could be considered in our study: First, although the use of BALB/c, hamster and guinea pig sera is acceptable, getting high volume of their serum is not possible at each blood sampling; second, it seems that the larger animals (dog and camel) have more economic logic due to getting high volume of their serum at each blood sampling but were not available and effective. Finally, according to the results of this research project it could be recommended to use sera from laboratory animals (hamster, BALB/c and guinea pig) for isolation and routine cultivation of Leishmania parasites, but FCS is recommended as a better choice in the projects in which a mass number of parasites are needed.

Conclusion

In this study, a rapid increase in parasite growth in media supplemented with hamster, BALB/c and guinea pig sera is considered. According to this finding, these laboratory animal’s sera could be recommended as a suitable alternative for FCS as it is an expensive and not readily available supplement in molecular biology studies.

Acknowledgement

This project was supported by the vice-chancellor of research, Shiraz University of Medical Sciences. The authors wish to appreciate Prof. M. Pourjafar, faculty of veterinary medicine, Shiraz University, Iran, for his kind cooperation in preparing the camel serum, and the authors thank the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for their assistance in English editing of this manuscript.

Compliance with ethical standards

Conflict of interest

The authors declared that there is no any conflict of interest.

Ethics approval

This work was approved by the ethics approval form (8668).

Footnotes

Publisher's Note

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