Advances in Infectious Diseases, 2013, 3, 231-237
Published Online December 2013 (
Open Access AID
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.
Received August 27th, 2013; revised September 26th, 2013; accepted October 3rd, 2013
Copyright © 2013 Alaa B. Ismael et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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
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
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
Open Access AID
Development of New Strategy for Non-Antibiotic Therapy: Dromedary Camel Lactoferrin
Has a Potent Antimicrobial and Immunomodulator Effects
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
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-
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
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-
Open Access AID
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
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
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
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
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
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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Has a Potent Antimicrobial and Immunomodulator Effects
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