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Host serum modifies the drug susceptibility of Babesia bovis in vitro

Published online by Cambridge University Press:  02 December 2004

S. BORK ,
M. OKAMURA ,
T. MATSUO ,
S. KUMAR ,
N. YOKOYAMA  and
I. IGARASHI
S. BORK
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
M. OKAMURA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
T. MATSUO
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
S. KUMAR
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
N. YOKOYAMA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
I. IGARASHI
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
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Abstract

Babesia parasites generally require a defined percentage of serum in the culture medium for their in vitro growth. In this study, we attempted to culture Babesia bovis in a serum-free condition. The growth pattern and morphology of B. bovis in serum-free (plain) GIT medium were unaltered as compared to those of the standard growth condition containing 40% bovine serum in M199. When exposed to the test drugs, the parasite in plain GIT medium showed clearly lower IC50 values than those in 40% serum-containing GIT medium, indicating that several serum components may interfere with the drug bio-availability. Therefore, the serum-free culture system is useful for standardizing drug test protocols and understanding the roles of serum factors in the drug test.

Keywords

Babesia bovisin vitro cultivationserum-free mediumdrug susceptibility

Information

Type
Research Article
Information
Parasitology , Volume 130 , Issue 5 , May 2005 , pp. 489 - 492
Copyright
© 2004 Cambridge University Press

INTRODUCTION

The microaerophilous stationary phase system (MASP) was first established as a successful method for the continuous in vitro cultivation of Babesia bovis (Levy & Ristic, 1980), which is a major aetiological agent of bovine babesiosis in many tropical and subtropical regions of the world (Kuttler & Young, 1984), and further improved by Goff & Yunker (1986). Subsequently, the MASP method has been expanded to the bovine Babesia bigemina and Babesia divergens (Vega et al. 1985; Gorenflot et al. 1991), and the equine Babesia equi and Babesia caballi (Holman et al. 1993, 1994; Avarzed et al. 1997). In the standard MASP culture method, Babesia parasites generally need a 40% volume of host serum in the appropriate media for their growth. However, the host serum represents inconsistent quality (Neves, 1991), which makes a direct comparison of results obtained from similar assays performed in different laboratories very difficult. Furthermore, the requirement for large quantities of serum has several restraints, such as high costs for the procurement and storage of serum (Ofulla et al. 1994), and difficulties in the isolation/purification of parasitic proteins in the presence of serum (James, Levy & Ristic, 1981).

To elucidate these problems, several serum-free cultivation systems have been established for B. divergens (Schrével, Grellier & Rigomer, 1992; Grande et al. 1997), B. caballi (Zweygarth, van Niekerk & de Waal, 1999; Ikadai et al. 2001), and Plasmodium falciparum (Asahi & Kanazawa, 1994). Recently, Jackson et al. (2001) successfully replaced the bovine serum by lipid-rich bovine serum albumin for several strains of B. bovis. In the present study, we have introduced the novel utility of a plain GIT medium for continuous in vitro serum-free culture of B. bovis. Subsequently, the serum-free culture system was evaluated by comparing with standard serum conditions in terms of efficacy of several babesiacidal drugs that had been demonstrated to inhibit the in vitro growth of B. bovis in the standard growth medium (Bork et al. 2003a,c, 2004).

MATERIALS AND METHODS

In vitro cultivation of B. bovis with various media

The Texas strain of B. bovis was routinely maintained in purified bovine erythrocytes using the standard growth medium consisting of medium M199 (Sigma-Aldrich, St Louis, MO, USA) and 40% bovine serum as described previously (Bork et al. 2003a). Infected erythrocytes were diluted with non-infected erythrocytes to obtain 1% parasitaemia in a 0·1 ml volume, and the mixture was subsequently suspended in 0·9 ml of the M199 or GIT medium (Nihon Pharmaceutical, Tokyo, Japan) supplemented with the indicated percentage of bovine serum. The suspension was cultured in 24-well culture plates (Nunc, Roskilde, Denmark). During the incubation period, the overlaid culture medium was daily replaced with 0·9 ml of the indicated fresh medium. Experiments were carried out in triplicate wells (for each medium) and in 3 separate trials to obtain conclusive results. Parasitaemia was determined daily as the percentage of parasitized erythrocytes (PPE) in Giemsa-stained erythrocyte smears by light microscopy. Differences in the percentage parasitaemia were statistically analysed by the independent Student's t-test, in which P<0·03 was considered to be of significant difference.

Comparative morphological studies of B. bovis cultured in plain GIT, plain M199, and 40% serum-containing M199 media were conducted by light microscopy as described previously (Bork et al. 2003a).

Drug tests and determination of IC50 values

The inhibitory capacities (expressed as IC50 values) of the following 5 compounds were evaluated on the growth of B. bovis in plain GIT medium as well as in 40% serum-containing GIT and -M199 media (the latter was regarded as a control): triclosan [5-chloro-2-(2,4 dichlorophenoxy)-phenol] (Sigma-Aldrich), clotrimazole (1-[(2-chlorophenyl) diphenylmethyl]-1H-imidazole), ketoconazole (cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazole-1-ylmethyl)-1,3-dioxolan-4-yl] methoxy] phenyl] piperazine) (both from Sigma Chemical), clodinafop-propargyl ((2R)-2-[4-[(5-chloro-3-fluoro-2-pyridinyl) oxy] phenoxy] propanoic acid-2-propynyl ester) (Riedel-de Haën Laborchemikalien, Germany), and heparin (Wako Chemicals Inc.). Experiments were carried out in triplicate for each drug concentration. The data obtained from 3 separate trials were combined and analysed to determine the IC50 values (Bork et al. 2003a,b,c, 2004).

RESULTS

In vitro cultivation of B. bovis in a serum-free medium

Fig. 1 (A) shows the in vitro growth patterns of B. bovis cultured in GIT and M199 media with or without various concentrations of bovine serum. The growth of B. bovis was optimal in the plain and 40% serum-containing GIT media and in the M199 media supplemented with 40 and 20% serum, showing PPE values of 15·8, 16·15, 16·3 and 14·6, respectively, after 3 days of cultivation, followed by M199 media with 10% (PPE 10·9) and 5% (PPE 6·3) bovine serum. M199 medium supplemented with less than 5% serum, and plain M199 did not support the growth, leading to degradation and death of the parasite. Moreover, B. bovis cultured in the plain GIT medium was successfully maintained for more than 12 months (data not shown).

Fig. 1. (A) In vitro growth rates of Babesia bovis in GIT and M199 media supplemented with different concentrations of serum in cultures for 3 days. Control: 40% bovine serum in M199; M199+20%, 10%, 5%: 20%, 10%, 5% bovine serum in M199; M199: plain M199; GIT: plain GIT. Asterisks indicate P values of <0·03. (B–D) Light micrographs of B. bovis, cultured in plain GIT (B), in M199 with 40% serum (C), and in plain M199 (D). Micrographs were taken on the third day of in vitro cultivation. Note the pycnotic appearance of the parasite in (D).

Morphologically, B. bovis cultured in the plain GIT (Fig. 1B) and 40% serum-containing M199 media (Fig. 1C) exhibited no differences in their characteristics in terms of size and shape as determined in light microscopy. In contrast, the parasites exposed to plain M199 medium (Fig. 1D), which did not support the serum, became degenerated, and died on the third day of cultivation.

Increased sensitivity of anti-babesial drugs in a serum-free medium

The growth of B. bovis was significantly inhibited in the presence of several drug concentrations in standard growth medium and in GIT medium with or without bovine serum. In order to determine the differences in the inhibitory capacity, the IC50 values of each drug compound was calculated and summarized in Table 1. The IC50 values of clotrimazole, triclosan, and ketoconazole were 7·3-, 6·1-, and 2·1-fold lower in the parasites cultured in the plain GIT medium than those cultured in 40% serum-containing GIT medium. Exposure to clodinafop-propargyl and heparin solely yielded slight reduction of the IC50 values of 1·9- and 1·5-fold, respectively, in the plain GIT medium. When comparing the IC50 values of the compounds in plain GIT or standard growth medium, CLT, KC and CP in standard growth medium were slightly lower (79, 73 and 86% of the value in plain GIT), while triclosan and heparin remained unaltered. As anticipated, the IC50 values of the drugs exposed to 40% bovine serum in M199 medium were virtually identical to those evaluated in previous reports (Bork et al. 2003a,b, 2004).

DISCUSSION

In the present study, the GIT medium was selected for the replacement of standard growth medium because of its widespread use as a multi-purpose medium for many cells (Ikadai et al. 2001). As a result, B. bovis was continuously propagated in the plain GIT medium without any supplementations. The growth patterns and the morphological appearance of B. bovis cultured in the plain GIT medium were also identical to those cultured in 40% serum-containing M199 medium, indicating that the GIT medium may provide an ingenious source as a basic culture medium for the parasite. Previously, Ikadai et al. (2001) also reported the successful cultivation of B. caballi in the plain GIT medium. Some components in the GIT medium seem to be essential to the parasite growth and maintenance. On the other hand, Jackson et al. (2001) cultured the B. bovis by using a lipid-rich bovine serum albumin, AlbuMAX™, as an alternative to bovine serum. Compared to our study, the average percentage of parasitaemia in their experiments was lower, and importantly, the costs for the ingredients were more than 10-fold higher. Further experiments will be required to identify the important growth factors, which can be used instead of host serum for the in vitro cultivation. Although Levy, Erp & Ristic (1981) reported the absolute requirement of 40% bovine serum in M199 for the successful growth of B. bovis, we demonstrated the unaltered propagation pattern of the parasite cultured in bovine serum as low as 20% in M199. A sufficient explanation of this interesting discrepancy is not possible and remains hypothetical, but it might be due to different parasitic strains used in the two studies, albeit Levy et al. (1981) do not mention the parasite's exact specification.

Several authors proved the usefulness of serum-free cultures in the drug susceptibility assays of P. falciparum as a reliable alternative standard laboratory practice (Ofulla et al. 1994), but similar information has been lacking in Babesia parasites. Here, we found that the IC50 values of B. bovis cultured in the plain GIT with triclosan, clotrimazole, ketoconazole, clodinafop-propargyl, or heparin were relatively lower as compared to those cultured in 40% serum in GIT or 40% serum in M199. This observation might be due to the presence of some serum components including a bovine serum albumin, which is considered to have higher affinity for any drugs, leading to increased IC50 values (Ofulla et al. 1994). The serum-free culture system used in the present study at least avoids taking into consideration the effects of serum factors on the results of drug tests. Further determination of the component(s) of GIT medium essential for the parasite propagation will provide not only a useful insight into the culture techniques but also a better understanding of the biology of Babesia species through the elucidation of the mode of antibabesial drug effects.

In conclusion, the serum-free in vitro culture method eliminates the serum effects, and thus will facilitate the standardization of drug test protocols among different laboratories.

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and by grants from the Special Coordination Funds for Science and Technology from the Science and Technology Agency and from The 21st Century COE Program (A-1), Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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Figure 0

Fig. 1. (A) In vitro growth rates of Babesia bovis in GIT and M199 media supplemented with different concentrations of serum in cultures for 3 days. Control: 40% bovine serum in M199; M199+20%, 10%, 5%: 20%, 10%, 5% bovine serum in M199; M199: plain M199; GIT: plain GIT. Asterisks indicate P values of <0·03. (B–D) Light micrographs of B. bovis, cultured in plain GIT (B), in M199 with 40% serum (C), and in plain M199 (D). Micrographs were taken on the third day of in vitro cultivation. Note the pycnotic appearance of the parasite in (D).

Figure 1

Table 1. IC50 values of different compounds in Babesia bovis