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Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin family protein. It is produced and secreted by mammary glands and neutrophils. One LF molecule can tightly and reversibly bind two Fe3+ atoms; thus, LF has Fe3+-free (apo) and Fe3+-bound (holo) states.1,2 Lactoferrin has broad-spectrum antimicrobial properties and acts in the primary defense against bacteria, viruses, fungi, and protozoa.3 It also has anti-protozoan activity against the intracellular form of Toxoplasma gondii,4,5 the intracellular form of Trypanosoma cruzi,6,7 and the intra-erythrocytic form of Plasmodium falciparum.8 The role of LF in defense against other types of protozoan-induced disease is poorly understood, but may involve various mechanisms.
Equine Babesia (Babesia caballi and B. equi), the latter of which has been recently reclassified as Theileria equi,9 are apicomplexan protozoa that infect the erythrocytes of horses and induce fever, anemia, jaundice, and edema. The transmission of these protozoa by ticks has been reported worldwide.10,11 The disease leads to substantial economic losses in the horse industry.12–14 During the asexual growth cycle in the natural host, equine Babesia protozoa live and multiply within erythrocytes. However, the growth cycle of these organisms is not fully understood. Understanding the basic molecular mechanisms involved in the asexual growth cycle, particularly those involved in the processes of growth and metabolism, may accelerate the development of effective therapeutic and preventive methods against equine Babesia infection. We examined the anti-babesial activity of four types of LF and determined whether these four LFs have different effects on the in vitro growth of B. caballi and B. equi.
United States Department of Agriculture strains of B. caballi and B. equi were maintained in horse erythrocytes in continuous cultures as previously described.15–17 The basic culture medium contained RPMI 1640 medium (Sigma-Aldrich, Tokyo, Japan) plus 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid for B. caballi and medium 199 (Sigma-Aldrich) plus 0.1 mM hypoxanthine (Invitrogen Corp., Carlsbad, CA) for B. equi. Horse erythrocytes at a hematocrit of 10% in RPMI 1640 medium or medium 199 supplemented with 40% (v/v) horse serum were distributed in a 24-well culture plate (1 mL of suspension/well) and cultured in a humidified atmosphere of 5% CO2 in air at 37°C; the culture medium was changed daily.
Bovine native-LF (~38% Fe3+ saturation) and bovine LF-hydrolyzate produced by pepsin cleavage (LF-H) were obtained from Morinaga Milk Industry Co. Ltd. (Zama, Japan). Iron-saturated (holo)-LF (~70% Fe3+ saturation) and iron-deficient (apo)-LF (0% Fe3+ saturation) were prepared according to the method of Law and Reiter.18 These LFs were dissolved in the culture medium and filter-sterilized through a 0.22 µm Millex filter unit (Millipore Co., Bedford, MA) before use.
The in vitro assay to measure growth inhibition was performed as follows. Babesia protozoa cultures that had reached 5–10% parasitemia were mixed with uninfected erythrocytes to obtain an initial parasitemia of 1%, and 100 µL of this suspension was mixed with 900 µL of the test medium in four replicas. The medium overlaying the erythrocytes was replaced with 900 µL of fresh medium containing the LF fraction appropriate to the treatment once every 24 hours. Thin smears were made, and the parasitemia was monitored to give approximately 1,000 erythrocytes for microscopic examination using Giemsa-stained blood smears. As a control, identical cultures were prepared without the addition of the LFs to the culture medium.
The concentrations that resulted in 50% inhibition (IC50) were estimated from data obtained on day 4 of the in vitro culture using a log-concentration/response probit analysis.19 Concentration values were transformed into logarithms, and percentage inhibition values were transformed into probits. The transformed data were processed using linear least squares regression analysis.20 Inhibitory concentrations were calculated according to the formula probit y = a + b logx, and were then transformed to the antilogx. Growth inhibition at a given LF concentration was calculated using the same formula; the logx was entered, and the resulting y was converted from probit percentage inhibition using a computer program. The differences in the percentage of parasitemia were analyzed using the independent Student’s t-test, with a P value < 0.01 representing a significant difference.
Babesia caballi and B. equi were grown in the in vitro cultures, starting from 1% parasitemia under the indicated concentrations of the various LFs, and the percentages of parasitemia were compared with values obtained from control cultures. The growth of B. caballi was significantly suppressed in the presence of 2.5 mg/mL and 5 mg/mL of apo-LF on day 4 of culture (Figure 1A
). The IC50 value for apo-LF was 2.7 mg/mL for B. caballi on day 4 of culture. However, B. caballi growth was not inhibited in the presence of 5 mg/mL native-LF, holo-LF, or LF-H on day 4 of culture. Similarly, growth of B. equi was not inhibited in the presence of 5 mg/mL native-LF, holo-LF, or apo-LF on day 4 of culture (Figure 1).
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, the inhibition of growth of B. caballi in the heparin-treated medium was similar at both concentrations (2.5 mg/mL and 5 mg/mL apo-LF-HP) as that in the corresponding concentrations of apo-LF. The IC50 value of apo-LF-HP against B. caballi was 2.4 mg/mL, which was similar to that of RPMI 1640 medium containing 5 mg/mL apo-LF. These results suggest that the inhibitory effect on the growth of B. caballi is not influenced by apo-LF.
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There may be several mechanisms responsible for the antimicrobial properties of LF. Lactoferrin may interfere with Fe3+ uptake by microbial pathogens by binding to the Fe3+ needed for growth.3,26,27 It has direct antimicrobial activity because it can bind the surface membrane of microbial pathogens via the action of lactoferricin (LFcin); pepsin treatment of LF generates stable antimicrobial peptides from the N-terminal region, which are active against bacteria, viruses, fungi, and protozoa.3 Moreover, LFcin has greater antibacterial activity than LF.3 Our data showed that apo-LF inhibited the growth of B. caballi but not B. equi, and that none of the four types of LF tested inhibited the growth of B. equi. The effect of the peptide produced by pepsin cleavage of LF-H appears to be different from the B. caballi inhibitory effect because LF-H did not inhibit growth of B. caballi. Fe3+ atoms were not detected in apo-LF, suggesting that a deficiency of Fe3+ in the culture medium was not involved. Moreover, the growth of the intra-erythrocytic form of P. falciparum was significantly inhibited by 30 µM (2.4 mg/mL) apo-LF and holo-LF.8 This is thought to result from the generation of oxygen free radicals by the LF/Fe3+ complex, which damages membranes of erythrocytes and protozoans.8 However, our data showed that growth of B. caballi was inhibited only by apo-LF. Therefore, it is suggested that the mechanism of action of LF in B. caballi differs from that in P. falciparum. The inhibitory effect of apo-LF on B. caballi appears to be related to inactivation or inhibition of a growth factor in the culture medium. Growth inhibition of B. caballi and B. equi did not appear to be related to general actions of LF or LFcin, suggesting that this was a novel antimicrobial mechanism of LFs.
In conclusion, we demonstrated that apo-LF inhibits the in vitro growth of B. caballi. Our data suggest the possibility of a novel mechanism of antimicrobial action of apo-LF. Further investigation of the growth inhibition caused by apo-LF will be helpful in understanding the growth factors needed by B. caballi. In particular, it will be important to identify whether different molecules are required by B. caballi and B. equi (T. equi) for metabolism and growth in blood.
Received September 15, 2004. Accepted for publication January 10, 2005.
Financial support: This study was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and the Kitasato University Research for Young Scientists.
* Address correspondence to Hiromi Ikadai, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. E-mail: ikadai{at}vmas.kitasato-u.ac.jp 
Authors’ addresses: Hiromi Ikadai, Nona Shibahara, Hiroko Tanaka, Noboru Kudo, and Takashi Oyamada, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. Tetsuya Tanaka and Kei-ichi Shimazaki, Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan. Aya Matsuu, Department of Small Animal Medicine, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. Ikuo Igarashi, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Reprints requests: Hiromi Ikadai, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan, Telephone: 81-176-23-4371, Fax: 81-176-25-0165, E-mail: ikadai{at}vmas.kitasato-u.ac.jp
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