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Archs oral BioL Vol. 38, No. 12, pp. 1057-1063, 1993 Printed in Great Britain. All rights reserved

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THE FUNGICIDAL EFFECT OF HUMAN LACTOFERRIN ON C,4NDIDA ALBICANS AND CANDIDA KRUSEI H. Nrrlwl,l'2 L- P. S.trr{.qRANAyAKE,r'* J. TnNovuo,3 K. M. Plt'lcr and T. H.q.N4ADA.2 roral Biology Unit, Prince Philip Dental Hospital, university of Hong Kong,-long Kong, 2Department

3Department of Cariology, ol Prosthetic Dentistry, Hiroshima University School of Dentistry, Japan and Finland of Turku, Institute of Dentistry, University (Accepted 27

* l-r

July 1993)

Summary-Five oral isolates each of Candida albicans and Candida krusei were studied for their sensitivity to the fungicidal effect of human lactoferrin. Significant inter- and intraspecies variations were observed and with most isolates the sensitivity ol C. krusei to lactoferrin was greater than that of C. albicans. Fungicidal activity of lactoferrin was dose-dependent and observable only with the iron-free form of the molecule (apo-lactoflerrin). Iron-saturated lactoferrin was ineffective against all isolates. Supernatant protein assJys and scanning electron microscopy indicated cell surface alterations-leakage of proteins ind formation of surface blebs-only in those Candida isolates that were sensitive to apo-lactoferrin. As lactoferrin is a common, non-immune, mucosal defence protein, its varying mode of action against C. albicans and C. krusei may be related to their different oral carriage rates. Key words: lactoferrin, Candida albicans, Candida krusei.

1981 ,, 1982;

INTRODUCTION

Candida albicans and related Candida species are the

most common opportunistic fungal pathogens

en-

countered in the mouth. Oral candidal carrtage and infection increase remarkably in patients with diseases, such as diabetes, immunosuppression and

xerostomia, and it is thought thaf the suppression of both local and systemic defence mechanisms may, at least in part, be responsible for such infestation

(Samaranayake, 1990). Human whole saliva contains a number of specific and non-specific defence factors. Amongst the nonspecific, lactoferrin, which is synthesized by acinar epithelial cells and also found in secondary granules of polymorphonuclear leucocytes, is recognized as an important mucosal defence factor (Reiter, 1983). In mucosal secretions, the lactoferrin molecule is pri-

marily iron-free (apo-lactoferrin), rarely

exceeding

20% iron saturation (Mazurier and Spik, 1980). There are many reports on the bacteriostatic or bactericidal effects of lactoferrin. Its bacteriostatic effects have been attributed to its high affinity for iron and consequent deprivation of this essential metal from pathogenic organisms (Cole et al., I97 6),, whereas the bactericidal effect, mediated only by apo-lactoferrin, is thought to be due both to iron deprivation and direct interaction with microbial cell walls (Arnold et al., 1982). Although the bactericidal effect of apo-lactoferrin has been characterized by many investigators (Arnold, Brewer and Gauthier, 1980; Arnold et al., *To whom all correspondence should be addressed. Abbreuiations: c.f.u., colony-forming units; SDS-PAGE,

sodium dodecyl sulphate-polyacrylamide gel electro-

phoresis.

Kalmar and Arnold, 1988), little is known

about the antifungal activity of apo-lactoferrin against Candida species. Arnold et al. (1980), for instance, have reported that the apo-lactoferrin was fungicidal for C. albicans, although iron-saturated

lactoferrin did not kill the organism. Subsequently others have confirmed the inhibitory effect of apolactoferrin on the growth of C. albicans, but not on C. krusei (Valentr et al., 1986). Recently, Soukka, Tenovuo and Lenander-Lumikari (1992) characterized in some detail the effect of apo-lactoferrin and partly and fully iron-saturated lactoferrin on C. albicans and demonstrated concentration-, time-, temperature- and pH-dependent fungicidal effects of apo-lactoferrin. However, they used only a single isolate of the organisms throughout. It is important to study interactions between lactoferrin and several isolates of Candida species as inter- and intraspecies variations in susceptibility to non-specific defence factors such as lysozyme have been described (Tobgi,

Samaranayake and MacFarlane, 1988). Accordingly we have now investigated the fungicidal effect of both apo-lactoferrin and iron-saturated lactoferrin against five oral isolates each of C. albicans and C. krusei. The relation between the fungicidal effects of apo-lactoferrin and the cell surface changes of these yeasts was also examined by scanning electron microscopy. MATERIALS AND METHODS

Micro -organisms and growth conditions

C. albicans GDH 16, 17, 18, L9 and 20, and C. krusei GDH 6,9, 13,23 and 24 wete used. All were oral isolates obtained from the routine microbiology services of the Glasgow Dental Hospital and School. All isolates were identified by sugar assimilation test using the API 20C system (API Products, Biomeruix, t057


1058

H. Nrrlwn et al.

Lyon, France) and 'the germ tube' test (Silverman et al., 1990); stock cultures were maintained at 4"C on Sabouraud dextrose agar (SDA; Oxoid Ltd, Basingstoke, U.K.). To prepare inocula for the assay, a loopful of each isolate was inoculated into brain-heart infusion broth (Oxoid Ltd, Basingstoke, U.K.), and grown aerobically at 37"C. After 18 h incubation, which corresponded to the exponential growth phase, the yeasts were harvested by centrifugation at 5000 g for 10 min and washed twice with ice-cold KCI buffer (0.05 ffiM, pH 7.0). The yeasts were resuspended in the buffer and standardized to a final concentration of about 5 x 106 c.f.u.lml using a spectrophotometer.

Because C. albicans is a dimorphic fungus the possible emergence of a hyphal phase was monitored

in all experiments. The cells remained in the

yeast

(blastospore) phase throughout the study.

Iron-free lactoferrin and iron -saturated lactoferrin Iron-free lactoferrin, which was purified from human colostruffi, was a generous gift from SynfinaOlefina Co., Brussels, Belgium. The purity and homogeneity of the preparations were analysed by fast protein liquid chromatography (LKB, 2138 Uvicord. S, Bromma, Sweden) and SDS-PAGE (PhastGel, PhastSystem, Pharmacra, Sweden) using

10-15% gradient gels. The iron-free state of the apo-lactoferrin was controlled by the ferrozine method as described by Soukka et al. (1992). The lactoferrin preparation was found to be practically lysozyme free (0.5 pglmg lactoferrin) when assayed

with Micrococcus diffusion plates (Lysozyme Kit, Kallestad Laboratories, Chaska, MN, U.S.A.) and with electrophoresis on SDS gels; the preparation was also free of IgA when tested by enzyme immunoassay.

Iron-saturated lactoferrin was prepared according to Mazurier and Spik (1980). In brief, apo-lactoferrin was dialysed against ferrous ammonium sulphate solution for 36 h at 4"C and the iron-saturated lactoferrin was subsequently dialysed against distilled water for another 36 h before the experiments. Fungicidal assay The antifungal effects of apo-lactoferrin and ironsaturated lactoferrin were examined according to the method described by Soukka et al. (1992) with some modifications. To determine the optimal assay conditions, 100 pl of apo-lactoferrin solution and 100 pl of C. albicans GDH20 suspension and phosph atebuffered KCI (0.05 mM; pH 7.0) were added into a sterile centrifugation tube to a final volume of 1.0 ml and to yield a yeast concentration of 5 x 106 c.f.u./ml.

The data from the dose-response study described above were used to compare the relative fungicidal effect of a standard apo-lactoferrin and iron-saturated lactoferrin concentration on five isolates each of C. albicans and C. krusei. This part of the study was done by incubating 100 p I of the yeast suspension in buffered KCI (containing approx. 5 x 106 c.f.u./ml) with 100 pl of either apo-lactoferrin or iron-saturated lactoferrin solution, for 150 min at 37"C (final concentration of apo-lactoferrin 20 pg/ml). Subsequently the viable yeasts in the suspension was assessed by spiral plating 100 p I of the assay suspension (diluted 1 :50) on to Sabouraud dextrose agar and estimating the resultant c.f.u. as described above. A suspension of 100 pl of the investigated Candida species in an equal volume of distilled water and made up to a total of 1.0 ml with KCI buffer was included as the control on each occasion the experiment was performed. A11 experiments were done on two separate occasions, with quadruplicate determinations on each

Protein assa)) and scanning

electron -microscopic

Immediately after each experiment the remainder

of the test and control samples was centrifuged at 1000 g for 10 min and the protein content of the supernatants determined with a protein assay reagent (Nikawa and Hamada, 1990). The approximate number of apo-lactoferrin molecules adsorbed per yeast cell was estimated in one experiment where apo-lactoferrin had no significant effect on the yeast viability. This was done by deducting the protein content in the supernatant from the total protein content (at the

beginning of the experiment) and dividing this figure by the total number of yeast cells in the suspension; the molar equivalent employed being l0 pg apo-lac-

- 0. 12 pM (Arnold et al., 1981). For scanning electron microscopy a small volume

toferrin

of the sample deposit was applied to glass strips precoated with gelatin and fixed with 2% osmium tetroxide for 4h, at 4"C. The fixed specimens were then freeze dried, sputter coated with a layer of gold to a thickness of 200-300 A, and observed in a JXA-840 scanning electron microscope (Joel, Tokyo, Japan)

.

,n

Statistical analysis

All the numerical data obtained were analysed by Student's /-test or analysis of variance at 5 and I% levels. RESULTS

-lactoferrin on Candida

carefully vortexed, 100 pl of each sample was diluted 1:50 and plated on Sabouraud dextrose agar using a spiral plater (Spiral Systems, Cincinnati, OH, U.S.A.)

irrespective of whether the incubation period was 45 or 150 min. Exposure to a 20 pglml concentration of lactoferrin for 150min at 37"C was chosen as the standard experimental condition for further studies with both C. albicans and C. krusei isolates.

48 h

*'

obseruations

Effect of

and the resultant c.f.u. quantified after

'.q

occasion.

The final concentration of phosphate ions in the buffered KCI suspension was 0.15 mM. In the control tube lactoferrin was replaced by an equal volume of buffer. The final concentrations of apo-lactoferrin solution used were 5, 10 and 20 pglml.These tubes were incubated at 37"C for 45, 90 and 150 min with gentle shaking. After incubation, the tubes were

incubation at 37"C.

t

apo

In the case of C. albicans GDH 20, the fungicidal effect of apo-lactoferrin was dose-dependent (p <0.05, two-way analysis of variance) but not time-dependent (Fig. 1). For instance, exposure to apo-lactoferrin concentration of 20 pglml could reduce the viability of the yeasts by approx. 50%

,'


Lactoferrin and Candida specles 150 min

90 min

45 min

1059

100

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fJ.

50

U

oo

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U)

-l

0

4.5

05t020

10

20

Protein concentration of supernatant

05t02a

051020

Concentration of apo l,F (pg/ml)

(ttelmt) Fig. 3. The relation between the protein concentration of the supernatant of assay solution and the survival rate of

Fig. 1. The effect of apo-LF (-lactoferrin) concentration and the incubation period (45, 90 and 150 min) on C. albicans GDH 20. Each value represents an experiment with quadruplicated samples and bars indicate one standard deviation.

C. albicans (GDH 16, 17, 18, 19,20; solid circles) and C. krusei (GDH 6, 9, 13, 23, 24; open circles) isolates. The protein content of the control solution was less than 0.5 pglml.

The results of the next series of experiments to

dependent fungicidal effect of apo-lactoferrin was also observed with C. krusei [Fig. 2(b)], the effect

relative potency of apo-lactoferrin in killing the five isolates each of C. albicans and C. krusei are shown in Figs 2(a) and (b), respectively. There were significant interspecies variations in the susceptibilities to apo-lactoferrin amongst both C. albicans and C. krusei isolates. For instance, C. albicans GDH 18 was the most sensitive, whereas C. albicans GDH 19 was not inhibited by apo-lactoferrin. The strainassess the

6.0

3 r&

u

being most apparent with C. krusei GDH 9 and least

with C. krusei GDH 24. Furthermore, when the overall sensitivity of C. albicans isolates were coffrpared with the C. krusei isolates , C. krusei tended to demonstrate increased sensitivity to the protein, although this difference was not significant. Thus the mean survival rates of the five isolates of C. albicans and C. krusei after exposure to apo-lactoferrin were 52 and 27%, respectively. When the effect of iron-saturated lactoferrin on five isolates each of C. albicans and C. krusei was studied, no significant fungicidal effect was observed irrespective of the iron-saturated lactoferrin concentration or

the exposure period (data not shown). 4.5

Protein assa)) and scanning

oo

electron -microscopic

obseruations

On regression analysis of the total protein content

3.0

16** 1J** 1g**

of the supernatant of the assay solution and the survival rate of apo-lactoferrin treated Candida 19

(44.s) (s8.6) (14.e) (8e.8)

6.0

t]

Controt

ffi

apo LI;

20** (s 1 .4)

a significant negative relation between these

Fig. 3). The protein content of the control solution was negligible (0.01-0.5 pglml). On computing the molecules of apo-lactoferrin adsorbed by the apo-lactoferrin-resistant C. albicens GDH 19, a figure of 1.0 ^.,9.0 x 106 apo-lactoferrin molecules was obtained. Scanning electron microscopy was done on C. GDH 18 and C. krusei GDH 9, which were the most sensitive to apo-lactoferrin. Surface blebs and bleb-like aggregates were observed on the cell surfaces of apo-lactoferrin-treated cells of both species when compared with controls (Fig. 4). Such topographical alterations were not observed on the cell surfaces of apo-lactoferrin-treated C. albicans GDH 19, which was highly resistant to the action of lactoferrin (data not shown) albicans

D

U

species,

two variables was observed (, - 0.739; p < 0.05;

4.5

oo

o

3.0

(r** 9** (2.1.e) (2.1)

l3**

23**

(23.2) (1s.6)

24** (74.3)

Fig. 2. The fungicidal effect of apo-LF (-lactoferrin) on (a) C. albicans rsolates (GDH 16, 17,18, 19 and20) and (b) C. krusei (GDH 6,9, 13,23 and 24). Each assay was made on two separate occasions with quadruplicated samples on each occasion. Two asterisks indicate a significant fungicidal effect (p <0.01) compared with the controls and values within parentheses indicate percentage survival rates.

DISCUSSION

Superficial infections both in the oral and vaginal mucosa are the common presentation of human candidosis (Odds, 1988). Although mucosal secretions contain a variety of defence factors such as


H. Nrrawt et al.

1060

secretory IgA, lysozyme, lactoferrin and peroxidases, their antifun gal actrvity has not been fully characterized. Hence, our main aim was to investigate the anticandidal activity of human lactoferrin using a number of oral yeast isolates belonging to C. albicans and C. krusei species. The reported concentration of lactoferrin in whole saliva ranges from 8.5 to 24 pglml and increases 10-15-fold in dental plaque fluid (Cole et al., 1981; Tenovuo, 1989). Lactoferrin is synthesized in salivary glands and is also concentrated in polymorphonuclear leucocytes. The lactoferrin molecule in secretions and polymorphonuclear leucocytes is pri-

marily iron-free (apo-lactoferrin rarely

exceeding

Arnold et al. (1987) demonstrated log 4 and log

2.5

in Strep. mutans concentrations, respectively, under similar experimental conditions. Although it is difficult to offer a reason for this disparity, the shear surface area of the yeast cells reduction

12.9-7.2 x 2.9-15.2 ttm (Odds, 1988)l compared with the much smaller surface area of streptococci (1 pm dia) may explain this phenomenon, particularly if the activity of apo-lactoferrin is mediated via cell surface adsorption. The fact that susceptibility to apo-lactoferrin was associated with increased concentration of proteins in the supernatant could be explained by the following. Apo-lactoferrin may have either specifically or non-

with cell

membranes and

20% saturation (Massons, Heremans and Schonne,

specifically interacted

re6e). C. albicans was chosen for the study as it was the most common oral Csndida species isolated whilst C. krusei was selected for its purported moderate virulence and its relatively low isolation rate from the mouth. However, C. krusei ts an increasingly com-

thereby increased the cell permeability, as recent data indicate that iron-binding proteins (apo-lactoferrin and transferrin) damage the outer membranes of Gram-negative bacteri a and alter the membrane permeability (Ellison, Giehl and LaForce, 1988). As it is also known that partly and fully iron-saturated lactoferrin exhibit both specific and non-specific binding to Gram-negative bacteria (Kalfas et al., l99I) the nature of apo-lactoferrin interaction with cell walls of

mon emerging pathogen, particularly in immunocompromised patients including those with human immunodeficiency virus infection (Samaranayake,

Candida remains

ree2).

to be determined. One way of

Our results in the first series of experiments with a single isolate of C. albicans confirm the findings of Soukka et al. (1992), who reported that the candidacidal activity of apo-lactoferrin is dose-dependent and not time-dependent, at pH 7 .0. The fact that the candidacidal activity was relatively constant, irrespective of the exposure period (Fig. 1), implies that once the total lactoferrin in the supernatant is exhausted-due possibly to interactions with fungal cell

elucidating the specificity of apo-lactoferrin binding would be to introdu ce a competing protein into the reaction system, although this was not done here. The apo-lactoferrin-induced cell surface changes we observed by scanning electron rnicroscopy give credence to the theory that lactoferrin may confer

walls-then the residual 'resistant'

zyme systems consequential to lactoferrin adsorption (Laible and Germaine, 1985). Furthermore, increased

structural changes on microbial cell walls. Alternatively, the candidacidal activity may be an indirect effect of the activation of intracellular autolytic en-

cells in the suspension are protected from the action of apo-lactoferrin. The other possibility of continued exposure to lactoferrin after plating out (a 'carry over' effect) can be ruled out as the yeast suspension was diluted 50-fold before inoculation. There were varying degrees of susceptibility to

metabolism, such as hydrogen peroxide or hydroxyl radical (OH), may be involved (Lassiter et al., 1987). In addition, the classic action of iron deprivation by lactoferrin is likely to have played an important

apo-lactoferrin between different C. albicans and C. krusei isolates. Thus, C. albicans GDH 19 and C. krusei GDH 24 were relatively resistant to the enzyme compared with their counterparts. Furthermore, a

demonstrated indirectly by the impotency of iron-saturated lactoferrin to tnteract witn- both C. albicans and C. krusei isolates. These data confirm the results

significant negative correlation between the increased survival rate of yeasts exposed to apo-lactoferrin and

the protein concentration

in the supernatant was

noted, tempting us to suggest that the candidacidal activity may be associated with effiux of proteins into the medium (Fig. 3). On further analysis, it was evident that even when the yeasts were resistant to the action of lactoferrin, as in the case of C. albicans GDH 19, the organisms adsorbed 1.0 ^.,9.0 x 106 molecules of apo-lactoferrin

per yeast. According to Arnold et al. (1982),

106

apo-lactoferrin molecules per target cell of Strepto' coccus mutans are adequate for the bactericidal ac' tivity of apo-lactoferrin. The extreme avidity of cationic proteins such as apo-lactoferrin towards microbial cell walls is well known, although how a selective yeast cell population may have resisted its action needs to be further studied. Furthermore, in our study only a 0.8-0.9 log reduction in yeast cell numbers was seen, even with the most susceptible isolates of Candida,,whereas Lassiter et al. (1981) and

generation

of

metabolic by-products

of

aerobic

part in the fungicidal activity of apo-lactoferrin,

as

of previous workers who found that iron-saturated lactoferrin had insignificant effect on C. albicans (Soukka et al., 1992). Another reason for the inactivity of iron-saturated lactoferrin is the conformational changes of the lactoferrin molecule that accompany iron saturation. Further, there afe other factors operating in uiuo that need to be considered in evaluating the suscepti-

bility of Candida to lactoferrin. For instance, although C. albicans is sensitive to peroxi-

dase/SCN/H2O, system (Lenander-Lumikari, 1992),

which

is one of the major non-immune

defence

systems in the mouth (Tenovuo and Pruitt, 1984), peroxidase-mediated inhibition of C. albicans ls comptetety blocked by in uiuo concentrations of salivary

phosphate (Leander-Lumikari, 1992). In addition, Soukka et al. (1992) demonstrated in uitro inhibition of apo-lactoferrin activity against C. albicans when the phosphate ion concentration was 1.0 mM, in a pH 7.0 buffer. However the fungicidal activity reappeared when the phosphate ion concentration


Lactoferrin and Candida species

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H. Nrrawd, et al.

r062

was reduced to 0.1 mM. Therefore, our results were not unexpected as the final phosphate concentration in the reaction system was 0.15 mM. The foregoing observations of Soukka et al. (1992) imply that salivary concentrations of phosphate ions [range I .5-25.0 mM (Ferguson, 1939) I may negate the candidacidal activity of apo-lactoferrin, although whether binding of apo-lactoferrin to yeast cells in uiuo is affected under these conditions remains to be determined.

The fact that C. krusei was almost 50% more sensitive to apo-lactoferrin than C. albicans tndicates that lactoferrin may confer a selective colonization

pressure in uiuo on different Candida species. In previous studies with lysozyme we have shown that, out of six species of Candida, C. albicans was most resistant to the enzyme and C. krusei the most sensitive (Tobgi et al., 1989). These observations, together with the current data, possibly reflect the

subtle variations

in

cell-wall composition between

various Candida species. Indeed, some have suggested that C. krusei should be reclassified into a different

genus based

on the ultrastructural and

chemical composition of the cell wall and coenzyme Q numbers

(Yamada and Kondo, I9l2) In clinical terms, the relatively high resistance of C. albicans to apo-lactoferrin may partly explain its high oral carriage in healthy individuals as opposed to the

infrequent isolation

of C. krusei (Samaranayake,

MacFarlane and Williamson, 1987). On the contrary, C. krusei are more frequently isolated from patients

with dysfunctional salivary systems such as in Sjogrens syndrome (MacFarlane and Mason, 1973) and cytotoxic therapy (Samaranayake et al., 1984). Clearly, further work that simulates the in uiuo conditions and with a larger number of clinical isolates belonging to different Candida species is required to confirm these observations and elucidate the intriguing microbicidal mechanisms of lactoferrin. Acknowledgement-Ihrs study was partly supported by the Sigrid Juselius Foundation (J.T.).

of the outer membrane of enteric gram negative bacterta

by lactoferrin and transferrin. Infect. Immun. 56, 277 4-2881

.

( 1 989) Salivary electrolytes In Hu*on Saliua: Clinical Chemistry and Microbiology (Ed. Tenovuo J.), Vol I, pp. 75-88. CRC Press, Boca Raton,

Ferguson D. B. FL.

Kalfas S., Andersson M., Edwardsson S., Forsgren A. and Naidu A. s. (1991) Human lactoferrin binding to Porphyromones gingiualis, Preuotella intermedia and Preuotella melaninogenica. Oral Microbiol. Immun. 6, 350-355.

Kalmar J. R. and Arnold R. R.(1988) Killing of Actino-

bacillus actinomycetemcomitans by human lactofercrn. Infect. Immun. 56, 2552-2557. Laible N. J. and Germaine G. R. (1935). Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides . Infect. Immun. 48, 720-728.

M. o.

Lassiter

Newsome

A. L.,

Sams

L. D. and Arnold

480-485.

Lenander-Lumikari M. (1992) Inhibition of Candida albicans by the peroxidase/SCN 1}J202 system. Oral Microbiol. Immun. 7, 315-320. MacFarlane T. W. and Mason D. K. (1973) Changes in the oral flora in Sjogren's syndrome. J. clin. Path. 27 , 416421. Massons

P. L., Heremans J. E. and Schonne E.

(1969).

Lactoferrin, an iron-binding protein in neutrophilic leucocytes. J. exp. Med. 130, 643-658. Mazurier J. and Spik G. (1980) Comparative studies of the iron-binding properties of human transferrins. Biochim. biophys. Acta. 718, 4248. Nikawa H. and Hamada T. (1990) Binding of salivary and serum proteins to Candida albicans in uitro. Archs oral

Biol.

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"

odds F. c. (1983) Candida and candidosis, Znd

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Bailliere Tindall, London. Reiter B. (1983) The biological significance of lactoferrin.

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Samaranayake L. P. (1990) Host factors and oral candidosis. In Oral Candidosis (Eds Samaranayake L. P. and MacFarlane T. W.), pp. 66-103. Wright, London.

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Taxonomrc

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The fungicidal effect of human lactoferrin on candida albicans and candida krusel