Development of New Strategy for Non-Antibiotic Therapy: Dromedary Camel Lactoferrin Has a Potent Antimicrobial and Immunomodulator Effects

doi:10.4236/aid.2013.34034

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Advances in Infectious Diseases, 2013, 3, 231-237

Published Online December 2013 (http://www.scirp.org/journal/aid)

http://dx.doi.org/10.4236/aid.2013.34034

Open Access AID

231

Development of New Strategy for Non-Antibiotic Therapy:

Dromedary Camel Lactoferrin Has a Potent Antimicrobial

and Immunomodulator Effects*

Alaa B. Ismael1,2#, Salama M. Abd El Hafez3,4, Manal B. Mahmoud4, Abdel-Kader A. Elaraby5,

Hany M. Hassan4

1Department of Medical Biotechnology, Faculty of Applied Medical Sciences, Taif University, Turaba, KSA; 2Department of Animal

Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt; 3Department of Medical Microbiology, Faculty of

Applied Medical Sciences, Taif University, Turaba, KSA; 4Immunobiology and Immunopharmacology Unit, Animal Reproduction

Research Institute (ARRI), Giza, Egypt; 5Holding Company for Biological Products & Vaccines VACSERA, Giza, Egypt.

Email: #[email protected]

Received August 27th, 2013; revised September 26th, 2013; accepted October 3rd, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The human and bovine lactoferrin have been studied extensively, but very few reports have been published concerning

camel lactoferrin (cLf). The present study aimed to isolate cLf and evaluate its efficiency including antimicrobial activ-

ity and immunomodulator effects. cLf isolation was attempted from camel milk whey using a cation exchange chroma-

tography by SP-Sepharose. The antimicrobial activity of the isolated cLf was investigated against Staphylococcus au-

reus (S. aureus), Streptococcus agalactiae (S. agalactiae), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P.

aerogenosa) strains. The immune effect of cLf was studied by lymphocyte transformation test. It was found that cLf

was separated around molecular weight of 80 kDa and showed significant inhibitory effect against E. coli followed by P.

aeruginosa, S. agalactiae and S. aureus. cLf increased lymphocyte transformations mean values in a dose dependant

manner. The highest transformations mean value was determined at 50 µg/mL. In conclusion, these results suggest that

cLf is a potent natural antimicrobial and novel immunomodulator agent.

Keywords: Dromedary Camel Lactoferrin; Isolation; Antimicrobial and Immunomodulator Effects

1. Introduction

Few studies have been reported on camels and camel

milk [1]. Dromedary camel milk and their products are a

good nutritional source for the people living in the arid

and urban areas. In addition, fresh and fermented camel

milk were reported to provide particular health benefits

to the consumer depending on the bioactive substances in

milk [2]. Antibiotics are commonly used for both pro-

phylaxis and treatment of various bacterial infections in

human and farm animals. In recent years, antibiotics re-

sistance in bacteria of animal origin and its impact on

human health have drawn much attention worldwide [3].

Bovine mastitis is the most common cause for the use of

antibiotics agents in lactating dairy cattle [4] and the de-

tection of antibiotics residues in milk poses health haz-

ards to consumers, and the cause of high economic impor-

tance because such milk is unfit for processing and subse-

quent consumption [5]. Moreover, the antibiotic therapy

has many complications as hypersensitivity, direct toxic-

ity, antibiotic-induced immunosuppresion and super-infec-

tions. This is highlighting the need for a new strategy for

non-antibiotic therapy using novel immunomodulators as

naturally released immunomodulators (Lactoferrin (Lf),

cathelicidins and defenses) or bacterial products (Perip-

lasmic proteins and lipopolysaccharides).

Lactoferrin (Lf), is an iron-binding glycoprotein found

in a variety of body secretions including tears, bronchial

mucus, and saliva and it is found in high concentrations

in the mammary secretions of nonlactating dairy animals.

It is important in regulation of iron metabolism [6]. This

natural antimicrobial agent is a multifunctional bioactive

molecule with a critical role in many important physio-

*No conflict of interest to declare.

#Corresponding author.

Development of New Strategy for Non-Antibiotic Therapy: Dromedary Camel Lactoferrin

Has a Potent Antimicrobial and Immunomodulator Effects

232

logical pathways. Lf could elicit a variety of inhibitory

effects against microorganisms, comprising stasis, cidal,

adhesion-blockade, cationic, synergistic, and opsonic me-

chanisms. Broad-spectrum activities against different bac-

teria, viruses, fungi, and parasites, in combination with

anti-inflammatory and immunomodulatory properties,

make Lf a potent innate host defense mechanism [7]. The

large potential applications of Lf have led scientists to

develop this nutraceutical protein for use in feed, food

and pharmaceutical applications.

Camel lactoferrin (cLf) purification, biochemical, and

immunological characterization have shown its similarity

to human and bovine Lf, as well as the cross-react with

the anti-human Lf antibodies [8-10]. The amounts of

lactoferrin and immunoglobulins were found to be great-

er in dromedary camel milk than bovine or buffalo milk

[8,10,11]. Incubation of human leukocytes with cLf leads

to a complete virus entry inhibition after seven days’

incubation. Thus, cLf markedly inhibits hepatitis C virus

genotype 4 infection of human peripheral blood leuko-

cytes [12]. The miR-214 is directly involved in Lf ex-

pression and Lf mediated cancer susceptibility (proapop-

totic activities) in mammary epithelial cells [13].

Many processing technologies have been developed to

isolate the high purity fraction of Lf. And most of the te-

chnologies use a cation exchange chromatography on

SP-Sepha-rose [14,15].

The aim of this investigation was mainly to isolate cLf

from camel milk whey and evaluate its efficacy in vitro

including antimicrobial and immunomodulator effects.

We use cLf but not bovine Lf because cLf is more bioac-

tive [16].

2. Materials and Methods

2.1. Isolation of Lactoferrin from Camel Milk

Whey

Lactoferrin (Lf) isolation was attempted from camel milk

whey. It was purified using a cation exchange chroma-

tography on SP-Sepharose following the procedure that

previously described [14]. Briefly, milk whey was ob-

tained from camel milk using ultra speed centrifuge,

15000 × g at 4˚C for 30 min. Skimmed milk was then di-

luted 1:1 with the dilution buffer (0.04 M NaH2PO4, 0.8

M NaCl, 0.04% (v/v) Tween 20, pH 7.4) and it was in-

cubated with SP-Sepharose at 4˚C overnight. Afterwards,

the SP-Sepharose was washed with the washing buffer

(0.02 M NaH2PO4, 0.4 M NaCl, 0.02% (v/v) Tween 20,

pH 7.4) to elude the unbound proteins. The gel then

packed into a column (5 × 30 cm or 3 × 30 cm, depend-

ing on the milk volume) and lactoferrin was eluted with

the elution buffer (0.02 M NaH2PO4, 1 M NaCl, pH 7.4).

The column was run at a flow rate of 3 mL/min.

2.2. Electrophoresis of Milk and Fractions

Containing Lactoferrin

Purity control and characterization of camel Lf (cLf) was

done using sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). Collected fractions of ca-

mel milk whey and broad range protein ladder (Fermen-

tra SM1841) were resolved in 12% polyacrylamide mi-

nigel-protein II electrophoresis cell (Bio-Rad). Samples

were diluted in sample buffer 2-mercaptoethanol (Sigma

Chemical Co.), boiled for 5 minutes before being loaded

in the gels and run at 70 volts for 3 hours. Gels were

stained with 1% Coomassie blue R-250 (Sigma Chemical

Co.), then distained at room temperature in 5% methanol

and 7.5% acetic acid with shaking for 30 minutes. The

different fractions were quantified using Bio-Rad GS 700

imaging densitometer molecular analysis software against

broad range marker [17].

2.3. Antimicrobial Activity Assays

Escherichia coli (E. coli), Pseudomonas aeruginosa (P.

aerogenosa), Staphylococcus aureus (S. aureus) and

Streptococcus agalactiae (S. agalactiae) isolates were

used to study the antimicrobial activity of cLf. The tested

microorganisms were kept in their specific soft agar.

Working cultures were obtained by growing the tested

isolates on their specific media. After an overnight incu-

bation, an isolated colony was transferred to 10 mL of

Mueller-Hinton broth (MHB, Difco Laboratories, Detroit,

MI) and incubated at 37˚C for 16 - 20 h. Final concentra-

tion of 1 × 106 CFU/mL was used. A volume of 1 mL of

cLf solution in different concentrations (1 and 3 mg/mL)

was added to 4 wells of tissue culture plates (NUNC. A/S,

Roskilde, Denmark) for each of tested microorganisms as

previously described [18]. The tested microorganisms in

phosphate buffer saline (PBS, 10 mM, pH 7.4) was used

a control. Plates were incubated at 37˚C. Aliquots were

removed after 1, 3, 6, 12, 24 hours and ten serially di-

luted, then plated at 37˚C on Mueller Hinton agar (MHA,

Difco Laboratories, Detroit, MI) to be counted after 48 h

incubation. Total aerobic bacterial count (TBC) of tested

microorganisms was done in which viable aerobic me-

sophlic bacteria were determined as previously described

[19]. All equipments used were either sterile new glass or

plastic to avoid iron contamination. All experiments were

repeated at least two times.

2.4. In Vitro Lymphocyte Proliferation Studies

Lymphocyte proliferation test using MTT (3-(4, 5-di-

methyl thiazol-2-yl) 2, 5-diphenyl tetrazolium bromide)

was performed [20] with modification. Briefly, heaprini-

zed calf blood samples were aseptically collected

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Development of New Strategy for Non-Antibiotic Therapy: Dromedary Camel Lactoferrin

Has a Potent Antimicrobial and Immunomodulator Effects

233

in sterile tubes. The separation of lymphocytes was done

by layering of blood in Ficol (2:1) and centrifuged at 400

× g at 4˚C for 30 minutes to give packed blood cells with

granulocyte, interface layer (which contain lymphocytes)

and upper plasma layer. The interface layer was carefully

aspirated using sterile glass Pasteur pipette, then placed

in sterile tubes containing 2 mL RPMI 1640 medium.

Cells were washed 3 times with RPMI 1640 medium by

centrifugation at 400 × g for 10 min at 4˚C. After the last

wash, the sediment lymphocytes were resuspended in 1

mL of RPMI 1640 medium containing 10% fetal calf

serum (FCS). RBCs contamination, if any, was removed

by the distilled water lysis method. Lymphocytes were

seeded in triplicate in flat-bottom 96-well micro titer

plates (Costar) at 1 × 106 cells per well in 150 µL of cul-

ture medium either alone or with various concentrations

of cLf (10 µg/mL, 20 µg/mL and 50 µg/mL) or 15 µg of

Phytohemagglutinin (PHA) control per mL. Another 100

μL of cell suspension was added to three sets of triplicate

wells of a RPMI-1640 containing different concentration

of cLf (10 µg/mL, 20 µg/mL and 50 µg/mL) plus 50 µL

PHA in conc. of 15 µg/mL. The plates were incubated

for 3 days under 5% CO2 at 37˚C. Then 100 μL of su-

pernatant was removed from the wells and 10 μL of MTT

solution was added to all the wells. The plate was incu-

bated further for 4 h at 37˚C. The MTT formazon was

extracted from the cells using dimethyl-sulphoxide (100

μL/well). Then the OD was taken using an ELISA reader

at a test wavelength of 570 nm. All experiments were

repeated at least two times.

2.5. Statistical Analysis

The Statistical Products and Service Solutions (SPSS)

program was used for all analysis [21]. Data were ex-

pressed as mean ± standard error (SE). Comparisons were

tested using an analysis of variance (ANOVA) test. A

difference was considered to be significant at P < 0.05.

3. Results

3.1. Isolation and Characterization of Camel

Lactoferrin

The results revealed that the cLf was separated around

molecular weight of 80 kDa (Figure 1).

3.2. Antimicrobial Effect of Camel Lactoferrin

The antimicrobial activity of the isolated cLf was inves-

tigated against Streptococcus agalactiae (S. agalactiae),

Staphylococcus aureus (S. aureus), Escherichia coli (E.

coli) and Pseudomonas aeruginosa (P. aerogenosa)

strains. The cLf showed significant inhibitory effect

against E. coli followed by P. aeruginosa, S. agalactiae

Figure 1. SDS-PAGE of various fractions of Lf purification

from camel milk whey. Lane 1, Molecular weight marker;

lane 2, Lf standard; lane 3-5, fractions eluted from SP-Se-

pharose.

and S. aureus (Table 1). The inhibition of growth by cLf

was concentration-dependent in which a significant in-

hibitory effect of E-coli was observed in a conc. of 1

mg/mL of cLf after 3 h and at conc. of 3 mg/mL after 1 h

of incubation. Severe inhibition of growth was observed

against P. aerogenosa and S. agalactiae at conc. of 3

mg/mL after 6 h and 12 h of incubation respectively. S.

aureus showed slight inhibition of growth at conc. of 3

mg/mL in compared to control.

3.3. Immunomodulator Effect of Camel

Lactoferrin

The immune effect of cLf was studied by lymphocyte

transformation test (LTT). Phytohemagglutinin (PHA)

was used as a control. The obtained results showed that

the lymphocyte transformation mean value of PHA was

2.37 ± 0.06 (Table 2 ). While the lymphocyte transforma-

tions mean values of cLf alone at concentrations of 10

µg/mL, 20 µg/mL and 50 µg/mL were 1.805 ± 0.040,

1.955 ± 0.045 and 2.39 ± 0.053 respectively (Table 2).

The cLf increased lymphocyte transformations mean va-

lues in a dose dependant manner. The highest transfor-

mations mean value was at concentration of 50 µg/mL.

On the other side, the lymphocyte transformation mean

values of cLf with PHA, at concentrations of 10 µg/mL,

20 µg/mL and 50 µg/mL were 2.12 ± 0.03, 1.941 ± 0.024

and 1.861 ± 0.1 respectively (Table 2). This means cLf

decreased lymphocyte transformations mean values in a

dose dependant manner.

4. Discussion

Lactoferrin (Lf), in this work, was isolated and purified

from camel milk whey using a cation exchange chroma-

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Development of New Strategy for Non-Antibiotic Therapy: Dromedary Camel Lactoferrin

Has a Potent Antimicrobial and Immunomodulator Effects

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Table 1. Antimicrobial effect of camel lactoferrin (cLf) on E-coli, P. aeruginosa, S. aureus and S. agalactiae counts after 1, 3, 6,

12, 24 hours of incubation.

Microbial count (CFU/mL) after

Items 1 hour 3 hours 6 hours 12 hours 24 hours

Control 49.000 370.000 2.9 × 106 3.1 × 107 2.7 × 107

cLf (1 mg/mL) 35.000 15.000 8000 500 CIG

E-coli count

cLf (3 mg/mL) CIG CIG CIG CIG CIG

Control 1.8 × 104 2.3 × 104 1.7 × 105 2.4 × 106 2.9 × 107

cLf (1 mg/mL) 142.000 111.000 43.000 21.000 17.000

P. aeruginosa

count

cLf (3 mg/mL) 107.000 93.000 17.000 950 950

Control 87.000 2.3 × 106 2.7 × 107 2.9 × 108 2.2 × 108

cLf (1 mg/mL) 73.000 2.1 × 106 2.6 × 107 2.7 × 108 2.1 × 108

S. aureus

count cLf (3 mg/mL) 56.000 1.7 × 106 2.0 × 107 2.1 × 108 1.9 × 108

Control 0.7 × 106 2.6 × 106 3.4 × 107 2.9 × 108 3.6 × 108

cLf (1 mg/mL) 0.4 × 106 1.8 × 106 2.3 × 105 1.8 × 104 2.1 × 105

S. agalactiae

count

cLf (3 mg/mL) 2.2 × 105 1.9 × 105 1.0 × 104 1000 3300

cLf: Camel lactoferrin; CIG: Complete inhibition of growth; N.B.: S. agalactiae was more diluted to be easily counted.

Table 2. Immunomodulator effect of camel lactoferrin (cLf) using lymphocyte transformation test (LTT).

PHA alone Camel lactoferrin alone Camel lactoferrin with PHA

Items 10 µg/mL 20 µg/mL 50 µg/mL 10 µg/mL 20 µg/mL 50 µg/mL

LTT means ± SE 2.37 ± 0.061.805 ± 0.040

(P < 0.05) 1.955 ± 0.045

(P < 0.05) 2.39 ± 0.053n.s 2.12± 0.03

(P < 0.01) 1.941 ± 0.024

(P < 0.001) 1.861 ± 0.1

(P < 0.001)

PHA: Phytohemagglutinin; n.s: non-significant.

tography on SP-Sepharose. Compared to the bovine spe-

cies, camel whey contains a higher content of antimicro-

bial factors such as lysozyme, lactoferrin and immu-

noglobulins [8-10]. Variation in the composition of whey

proteins from camel (Camelus dromedarius) colostrum

and milk was recorded [22] and shown to be rich in pro-

tective proteins, especially lactoferrin, peptidoglycan rec-

ognition protein and immunoglobulins IgG2 and IgG3.

Due to Lf large potential applications, many processing

technologies have been developed to isolate high purity

fractions. Cation-exchange chromatography is already

used for the production of Lf at industrial scale [14,16].

This technology has the advantage of producing Lf with

a high degree of purity (>90% dry basis). The limitation

of this technology for large-scale applications lies with

its high cost and its relatively low yield [23]. Characteri-

zation of camel Lf (cLf) was done using reduced poly-

acrylamide gel electrophoresis (SDS-PAGE). cLf was

separated around molecular weight of 80 kDa. However,

affinity membranes with immobilized triazinic dyes have

not achieved yet good acceptance in the biotechnological

industry, mainly because of their low capacity for pro-

teins in comparison with the same legends immobilized

on soft gels [24] and the dye leaching in the elution and

regeneration steps [25]. Although, under equilibrium con-

ditions, membranes show an acceptable chromatogra-

phic performance for Lf purification from bovine colos-

trums, better than the obtained with d-Sepharose, as a

model of soft gels [26], the main problems affecting in-

dustrial utilization of adsorptive dye membranes, such as

low capacity, dye leaching and pressure drop along the

fiber axis need to be overcome. On the other side, the

recovery of Lf from whey is a relatively difficult task,

because not only the huge volume of whey needs to be

dealt with, but also the major proteins complicate the se-

paration process [27].

The cLf showed significant inhibitory effect against E.

coli followed by P. aeruginosa, S. agalactiae and S. au-

reus. One of the first antimicrobial properties discov-

ered for Lf was its role in sequestering iron from bacte-

rial pathogens as in case of S. aureus [28] which is

known to be resistant to antimicrobials. It was later dem-

onstrated that Lf’s bactericidal function has been attrib-

uted to its direct interaction with bacterial surfaces [29]

and through an iron-independent mechanism [30] as in

case of E. coli [31]. Biofilm formation, which was pro-

Development of New Strategy for Non-Antibiotic Therapy: Dromedary Camel Lactoferrin

Has a Potent Antimicrobial and Immunomodulator Effects

235

posed as a colonial organization adhesion method for P.

aeruginosa, is a well-studied phenomenon. Through bio-

film formation, bacteria become highly resistant to host

cell defense mechanisms and antibiotic treatment [32]. It

is well known that some bacterial strains require high le-

vels of iron to form biofilms. Thus, Lf’s function as an

iron chelator has been hypothesized to effectively inhibit

biofilm formation through iron sequestration [33]. Occur-

rence in various milieus strongly emphasizes the signifi-

cance of the structure-function relationship in the multi-

functionality of the Lf [7].

Regarding the immune effect of cLf, the cLf increased

lymphocyte transformations mean values in a dose de-

pendant manner. The highest transformations mean value

was of lactoferrin in conc. of 50 µg/mL. This finding was

agreed with [34] who reported that the addition of re-

combinant human lactoferrin (Talactoferrin Alfa (TLf))

to human peripheral blood or monocyte-derived dendritic

cell cultures resulted in cell maturation, as evidenced by

up-regulated expression of CD80, CD83, and CD86, pro-

duction of proinflammatory cytokines, and increased ca-

pacity to stimulate the proliferation of allogeneic lym-

phocytes. In addition, this finding was agreed to some

extend with [35] who found that the effects of Lf in ex-

perimental models were differential and dependent on an

individual PBMC reactivity, mitogen or alloantigen and

Lf concentration. Generally, lymphocytes from donors

responsive to Lf exhibited higher proliferation indices to

PHA when compared with non-responsive individuals,

suggest that the differential action of Lf might be due to

its ability to sense the activation status of lymphocytes,

although he mentioned that data on Lf effects on mito-

gen-induced proliferation are scarce, though fairly con-

sistent both in the mouse and human systems. It has been

demonstrated that human and bovine lactoferrin inhibit

proliferative responses in vitro.

In addition, the cLf decreased lymphocyte transforma-

tions mean values in a dose dependant manner when

combined with PHA. This opinion goes hand in hand

with [36] who reported that purified lactoferrin, isolated

from human milk, was tested for its effect on human T-

lymphocyte proliferative responses to Phytohemagglu-

tinin (PHA) and to alloantigen in mixed lymphocyte cul-

ture. Lf inhibited proliferation in both assays in a dose-

dependent manner. The suppressive effect was not due to

Lf mediated cytotoxicity since washing cells that had

been pre-incubated with Lf restored their proliferative ac-

tivity. Lf was most effective in suppressing the PHA re-

sponse when added within 24 h of culture initiation. Iron

saturated Lf failed to inhibit PHA-induced proliferation,

suggesting that the mechanisms of suppression involve

the chelating property of Lf. The suppressive effect of Lf

on T-lymphocyte proliferative response in vitro supports

the notion that Lf has significant immunoregulatory po-

tential in vivo. The same agreement was concluded by

[35] that the effects of Lf on the proliferative response of

lymphocytes to PHA were generally stimulatory at lower

and inhibitory at higher concentrations of Lf. The increas-

ed production of cytokines may play a significant role in

the down-regulation of mitogen-induced lymphocyte pro-

liferation in the presence of Lf.

5. Conclusion and Recommendation

In conclusion, these results suggest that cLf is a potent

natural antimicrobial and novel immunomodulator agent.

The extensive uses of Lf in the treatment of various in-

fectious diseases in animals and humans have been the

driving force in Lf research, however, a lot of work is

required to obtain a better understanding of its activity.

Further studies will be needed for molecular cloning, pro-

moter analysis and identification of camel lactoferrin

gene.

6. Acknowledgements

This research was financially supported by the dean of

Scientific Research (Project No.1-433-1582), Faculty of

Applied Medical Sciences (Turaba), Taif University, KSA.

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