20

Dental Journal
(Majalah Kedokteran Gigi)

2020 March; 53(1): 20–23

Research Report

The potency of Immunoglobulin Y anti Porphyromonas gingivalis 
to inhibit the adherence ability of Porphyromonas gingivalis on 
enterocytes

Nova Andriani Hepitaria, Indeswati Diyatri, Markus Budi Rahardjo and Rini Devijanti Ridwan
Department of Oral Biology,
Faculty of Dental Medicine, Universitas Airlangga,
Surabaya – Indonesia

ABSTRACT
Background: Pophyromonas gingivalis (P. gingivalis) bacteria are the main type of bacterium that cause chronic periodontitis. 
Immunoglobulin Y (IgY) is a type of immunoglobulin found in poultry, such as chickens and birds. IgY can be used as an alternative 
method of preventing the accumulation of plaque that causes chronic periodontitis. Purpose: To determine the ability of IgY anti P. 
gingivalis to inhibit adherence of P. gingivalis. Methods: The samples were divided into eight groups, each group containing 10 ml of 
IgY anti P. gingivalis and 50 ml of enterocyte cells. The control group contained 50 ml of IgY anti P. gingivalis, and 50 ml of enterocyte 
cells. Serial dilution was carried out to the first seven groups, with the first group containing 90 ml phosphate-buffered saline (PBS) 
and 10 ml IgY anti P. gingivalis, and the second to seventh groups containing 50 ml PBS before adding 50 ml of enterocyte cells and 
50 ml of bacterial suspension per group. The number of bacteria was calculated as an adherence index value using a light microscope. 
Results: This study shows that IgY anti P. gingivalis significantly reduces the adherence index value of P. gingivalis. Conclusion: IgY 
anti P. gingivalis has potency to inhibit the adherence of P. gingivalis.

Keywords: adherence; egg yolk; IgY; Pophyromonas gingivalis

Correspondence: Rini Devijanti Ridwan, Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga. Jl. Mayjen. 
Prof. Dr. Moestopo 47 Surabaya 60132, Indonesia. Email: rini-d-r@fkg.unair.ac.id

INTRODUCTION

Periodontal disease or periodontitis is a bacterial infectious 
disease characterised by continuous inflammation, 
connective tissue damage, and alveolar bone destruction.1 
Severe periodontitis can lead to tooth loss. This disease can 
be found in approximately 5–20% of adults worldwide. 
Periodontal disease is divided into three types: aggressive 
periodontitis; chronic periodontitis; and periodontitis caused 
by systemic disease manifestation.2 The bacterium that 
causes chronic periodontitis is Pophyromonas gingivalis 
(P.gingivalis).3 This bacterium is a rod-shaped anaerobic 
Gram-negative bacterium. P. gingivalis bacteria inhabit the 
subgingival area. Some virulence factors of P. gingivalis 
bacteria include adhesives, capsules, lipopolysaccharides 
(LPS), proteases, and outer membrane proteins. Capsules 
can reduce the phagocytic activity for invasion; LPSs, 

protease enzymes and membrane proteins can aid bacterial 
aggregation on the cell surface and fimbriae.4

Pulling activity between the surface of the bacteria and 
the surface of the host cell is called bacterial adherence 
activity. There are three stages of bacterial adherence to the 
surface, namely transport, initial adherence (usually called 
bioattachment) and colonisation.5 Antibiotics can be used 
to kill bacteria, but antibiotics also have a negative effect, 
including the resistance or increased ability of bacteria to 
stay alive in the presence of antibiotics.6

Immunoglobulin Y (IgY) technology is an innovative 
technology that involves the non-invasive production 
of polyclonal antibodies from egg yolk, and due to 
its non-invasive nature, IgY technology has provided 
new opportunities in both therapeutic and prophylactic 
applications in human and veterinary medicine.7 IgY 
in the form of polyclonal antibodies is used as passive 

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v53.i1.p20–23

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23


21Hepitaria, et al./Dent. J. (Majalah Kedokteran Gigi) 2020 March; 53(1): 20–23

immunisation, and these antibodies derive from egg yolk, 
colostrum, or concentrated cow’s milk.8 IgY has the same 
biological role as IgG in mammals, as a major type of 
immunoglobulin that provides defence against infectious 
agents.9 IgY can be used to prevent periodontitis by 
inhibiting bacterial adherence to the cell surface, inhibiting 
enzyme activity, and neutralising the toxins produced by 
the periontopathogen.10 The aim of this research was to 
determine the ability of anti IgY of P. gingivalis in egg yolk 
to inhibit the adherence of P. gingivalis bacteria.

MATERIALS AND METHODS

This study was a laboratory in vitro experimental study 
with a post-test-controlled group design. IgY specific P. 
gingivalis was obtained from chicken eggs that had been 
injected with P. gingivalis (ATCC 33277) by as much as 1.5 
x 109 bacterial colony, three times a week for three weeks. 
In the fourth week, the egg yolks were taken. The sample 
used was a mixture of enterocyte cells and IgY that had been 
induced with P. gingivalis (ATCC 33277) bacteria, and the 
control was a mixture of enterocyte cells with P. gingivalis 
(ATCC 33277) bacteria with three times replication.

This research required the following research tools: 
a centrifuge, an anaerobic jar, a shaking incubator, a 
measuring cup, a petri dish, a light microscope with 1000x 
magnification, a glass slide, a Falcon tube, a microcentrifuge 
tube, a micropipette, a research subject, i.e. mice with body 
weight 135 g, specific P. gingivalis (ATCC 33277) IgY 
serum, a culture of P. gingivalis (ATCC 33277) bacteria, 
phospate buffer saline (PBS), Mueller-Hinton broth (MHB), 
a solution containing PBS pH 7.4 + 1 mm dithiothreitol 
(DTT), a solution containing PBS pH 7.3, a solution 
containing PBS pH 7.4 + 1.5 mg Ethylenediaminetetraactic 
acid (EDTA) + 0.771 mg DTT, methanol, violet crystals, 
safranin, lugol, and alcohol. One colony of P. gingivalis 
(ATCC 33277) that had been grown in blood agar, was put 
into a test tube containing brain heart infusion (BHI) media 
using an oese and incubated in an anaerobic atmosphere using 
a gas generating kit for 24 hours at 37°C. After the incubation 
period, an equal concentration of bacteria was processed in 
another test tube containing BHI media, so that it was to the 
same McFarland standard 0.5 (1.5 x 108 CFU/ml).11

Isolation of enterocytes was performed using the 
Weisler method.12 Enterocytes were taken from the small 
intestine of 6–8-week-old mice weighing 135 g. The mice 
were humanely destroyed and then dissected to remove 
a part of the small intestine. The small intestine that had 
been taken from the body of the mice was then cut across 
and minced into small pieces, then washed using a solution 
containing PBS pH 7.4 + 1 mm DTT, to remove dirt and 
mucus. After the intestinal tissue was clean, it was put 
into a falcon tube, then as much as 20 ml of the solution 
containing PBS pH 7.3 was added. It was then put into 
a water heater at 37oC and shaken with a shaker for 30 
minutes. The discarded supernatant was then replaced with 

as much as 30 ml of a solution containing PBS pH 7.4 + 
1.5 mg EDTA + 0.771 mg DTT, and was shaken using a 
water bath for 30 minutes at 37°C. After being shaken, 
the supernatant was removed. Falcon tubes containing 
enterocyte cells were washed using PBS. Next, they were 
left until the enterocytes had all settled at the bottom of the 
tube. After the enterocytes had settled, the supernatant was 
removed, then 20 ml of PBS was added, and the tubes were 
inserted into the microcentrifuge tube and centrifuged at 
1500 rpm for three minutes. 

Separation of the P. gingivalis IgY concentration 
was carried out using the dilution series method on the 
microcentrifuge tubes. The concentrations made were 1/10, 
1/20, 1/40, 1/80, 1/160, 1/320, 1/640, and these were put into 
Eppendorf tubes. Each tube was added 50 ml PBS solution 
except in a tube with a concentration of 1/10, in other six 
samples PBS solution was added as much as 90 µl and 10 
µl IgY P. gingivalis, then all of the samples homogenised 
using vortex. After that, 50 µl of the homogeneous solution 
was taken from the tube with a concentration of 1/10 using 
a micropipette and put into the tube with a concentration of 
1/20, after which it was homogenised using a vortex. The 
same procedure was carried out until the concentration 
reached 1/640. In the tubes with a concentration of 1/640, as 
much as 50µl of the solution was removed.13 For adherence 
to the test procedure, cultures of bacteria were centrifuged 
at 6000 rpm and 4oC for 15 minutes. The precipitate 
was suspended in PBS containing 1% of bovine serum 
albumin (BSA). The bacterial content used was 108/ ml. 
Then 50 µl of the enterocyte suspension was added to each 
concentration and shaken using a shaker in the water bath 
at 37oC for 30 minutes. As much as 50 µl of the mixture 
was then added to the bacterial suspension (108/ml) The 
mixture was incubated in the shaking incubator for 30 
minutes at 37oC. It was then centrifuged at 1500 rpm and 
4oC for 3 minutes, then as much as 100 µl of the liquid 
was disposed of.

Each precipitate was taken and smeared on a glass slide 
and painted with Gram stain. The glass slides were observed 
under a light microscope with 1000x magnification, and the 
number of bacteria attached to enterocytes were counted, 
being calculated for each observation of 100 enterocytes.14 
The Kruskal Wallis difference test was carried out in the 
control group and the treatment group to determine if the 

Table 1. Mean and standard deviation of the power of adherence 
of sample groups to P. gingivalis

Adherence Pg

N Mean Std. Deviation
Control 6 12.7583 6.44508
A-1/10 6 4.0733 .53638
B-1/20 6 5.3417 .85873
C-1/40 6 5.4517 1.75848
D-1/80 6 4.8500 1.77213
E-1/160 6 6.2950 1.89505
F-1/320 6 4.1700 1.58865
G-1/640 6 9.1000 4.19729

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v53.i1.p20–23

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23


22 Hepitaria, et al./Dent. J. (Majalah Kedokteran Gigi) 2020 March; 53(1): 20–23

significance value was below 0.05 (p<0.05), i.e. showed 
significant differences between groups. Then the Bonferroni 
test was carried out.

RESULTS

The treatment group with P. gingivalis (ATCC 33277) of 
concentration 1/640 had the largest mean of 9.1, whereas 
the treatment group with P. gingivalis (ATCC 33277) of 
concentration 1/10 had the smallest mean of 4.07.

The mean and standard deviation of the power of 
adherence in each treatment group of the P. gingivalis 
bacteria can be seen in Table 1. The research data were 
analysed using the Kruskall Wallis test. The results of 
the Kruskall Wallis test in this study were p = 0.000 (p 
<0.05), so it could be interpreted that there were significant 
differences between the data of the entire group.The 
Bonferroni test results, shown in Table 2, obtained a value 
of 0.00 between the control group and the sample groups, 
except in the group 1/320. This shows that there was a 
significant difference between the control group and the 
sample groups. Comparison between the sample groups 
produces a value of 1.000. This can be interpreted as there 
being no significant difference between the sample groups. 
Figure 1 shows the result of the experiment in adherence 
of bacteria onto the enterocyte cells.

DISCUSSION

IgY has several advantages compared to antibiotics, 
vaccines, and immunotherapy. The advantages of IgY 
compared to antibiotics are: 1) it is natural; 2) it is safe and 
is not absorbed into the body circulation (no toxic tissue 
residues); 3) it avoids environmental contamination with 
synthetic chemical drugs; 4) it does not induce specific 
pathogenic resistance of microorganisms since it is directed 
to multi epitopic antigenic targets that need multiple genes 
for their synthesis; 5) it is highly specific in its reactivity 
and controls only targeted pathogens without affecting 
normal bacterial flora; 6) it has a potentially broad spectrum 
of specificity when customised against viruses, bacteria 
or fungi; and 7) it does not induce adverse side effects 
as synthetic drugs do.10 The advantage of IgY as passive 
immunotherapy is that IgY has rapid action and high 
specific activity; it can be given to all ages ranging from 
infants to adults, including babies with low birth weight 
(LBW), patients with immunodeficiency, and pregnant 
women. It is not toxic and can be stored for a long time. IgY 
is attractive for oral immunotherapy because some of its 
properties can be taken from animals without hurting them, 
the binding to antigens is stronger than in mammalian IgG, 
and it reacts more to the same antigen. IgY is also a natural 
ingredient that does not cause side effects when taken orally 
unless the recipient has an allergy to eggs.15

Based on the results and analysis of the data, it was 
found that P. gingivalis IgY can inhibit the adherence 
of P. gingivalis bacteria. This is likely because IgY is a 
polyclonal antibody that can capture various epitopes on the 
cell surface of the bacterium. Antibodies are host proteins 
found in plasma and extracellular fluids that serve as the 
first response and comprise one of the principal effectors 
of the adaptive immune system. They are produced in 
response to molecules and organisms, which they ultimately 
neutralise and/or eliminate. The ability of antibodies to bind 
to an antigen with a high degree of affinity and specificity 
has led to their ubiquitous use in a variety of scientific and 
medical disciplines. The formation of antibodies is not 
only due to the binding of the epitope with the antibodies, 

 

B 

A 

Figure 1. Bacterial adherence in the sample group to P. 
gingivalis (A) in enterocyte cell. (B) control group. 
Arrow indicates P. gingivalis adherence to the 
enterocyte cell.

Table 2. Results of Bonferroni test to P. gingivalis

Groups 1 2 3 4 5 6 7 8
1
2 .000*
3 .003* 1.000

4 .004* 1.000 1.000

5 .001* 1.000 1.000 1.000

6 .017* 1.000 1.000 1.000 1.000

7 .000* 1.000 1.000 1.000 1.000 1.000

8 1.000 .173 1.000 1.000 .532 1.000 .200
Note: 1) control group; 2) treatment group 1/10; 3) treatment group 1/20; 4) treatment group 1/40; 5) treatment group 1/80; 6)
treatment group 1/60; 7) treatment group 1/320; 8) treatment group 1/640.
*There are significant differences between sample groups.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v53.i1.p20–23

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23


23Hepitaria, et al./Dent. J. (Majalah Kedokteran Gigi) 2020 March; 53(1): 20–23

but because bacteria are one type of antigen that has 
virulence factors that can stimulate antibody formation.16 
The main mechanism of IgY is binding to components on 
the surface of bacteria, such as outer membrane proteins, 
lipopolysaccharides,as well as colonisation tools such as 
vesicles and fimbriae which are virulence factors of the P. 
gingivalis bacteria.17 The adherence value of P. gingivalis 
bacteria is significantly different between the treatment 
groups and the control group because of the possibility 
that this bacterium has expressed its virulence factor, 
particularly adhesion.18

Fragments of antigen binding (FAB) possessed by 
IgY anti P. gingivalis can bind and recognise proteins on 
the cell surface of the bacterium. FAB from IgY anti P. 
gingivalis can also bind to fimbriae, which are bacterial 
movement tools, to attach to periodontal tissue and carry 
out colonisation activities. As shown in this study, IgY 
binds to enterocytes.19 The presence of fimbriae is a 
virulence factor that plays a major role in the adherence 
activity of the P.gingivalis.20 On periodontal tissue, 
periodontitis carried out by imbriae from this bacterium 
are bound by IgY P.gingivalis,21 whereas to carry out 
pathogenic activities, the process that must be carried on is 
that bacteria must be attached to the periodontal tissue and 
then carry out colonisation activities with similar bacteria.21 
Comparing concentration groups, based on the results of 
the data analysis, there were significant differences in the 
comparison of the control group with the concentration 
of the P. gingivalis, while in the 1/640 dilution group 
no significant difference could be seen, indicating IgY’s 
ability to inhibit bacterial adherence on enterocytes at a 
minimum concentration. However, comparing between 
fellow treatment groups, there was no significant difference. 
This shows that, to inhibit P. gingivalis bacteria, it was 
sufficient to use a minimum concentration of IgY anti P. 
gingivalis in this study (1/320). The conclusion of this study 
is that IgY anti P. gingivalis has the potency to inhibit the 
adherence of P. gingivalis on enterocytes, and this potency 
is not dependent on the concentration.

REFERENCES

 1.  Vargas Segura AI, Ilyina A, Segura Ceniceros EP, Silva Belmares 
Y, Méndez González L. Etiology and microbiology of periodontal 
diseases: a review. African J Microbiol Res. 2015; 9(48): 2300–6. 

 2.  Aljehani YA. Risk factors of periodontal disease: review of the 
literature. Int J Dent. 2014; 2014: 1–9. 

 3.  Ismail AD. Oral bacterial interactions in periodontal health and 
disease. J Dent Oral Hyg. 2014; 6(5): 51–7. 

 4.  How KY, Song KP, Chan KG. Porphyromonas gingivalis: an 
overview of periodontopathic pathogen below the gum line. Front 
Microbiol. 2016; 7: 1–14. 

 5.  Sandle T. Bacterial adhesion: an introduction. IVT Nerwork. 2013; 
: 1–8. 

 6.  Nami Y, Haghshenas B, Abdullah N, Barzegari A, Radiah D, Rosli 
R, Khosroushahi AY. Probiotics or antibiotics: Future challenges in 
medicine. J Med Microbiol. 2015; 64(2): 137–46. 

 7.  Baloch AR, Zhang XY, Schade R. IgY Technology in aquaculture - a 
review. Rev Aquac. 2015; 7(3): 153–60. 

 8.  Ba cht ia r E W, A fd ha l A , Meidyawat i R , So ejo e dono R D, 
Poerwaningsih E. Effect of topical anti-Streptococcus mutans IgY 
gel on quantity of S. mutans on rats’ tooth surface. Acta Microbiol 
Immunol Hung. 2016; 63(2): 159–69. 

 9.  Munhoz LS, Vargas GDÁ, Fischer G, Lima M de, Esteves PA, Ḧbner 
S de O. Avian IgY antibodies: characteristics and applications in 
immunodiagnostic. Cienc Rural St Maria. 2014; 44(1): 153–60. 

10.  Rahman S, Van Nguyen S, Icatlo FC, Umeda K, Kodama Y. Oral 
passive IgY-based immunotherapeutics: A novel solution for 
prevention and treatment of alimentary tract diseases. Hum Vaccines 
Immunother. 2013; 9(5): 1039–48. 

11.  Wicaksono AW, Setiawatie EM, Rubianto M. Daya hambat ekstrak 
buah mengkudu (Morinda citrifolia L.) terhadap pertumbuhan 
bakteri Porphyromonas gingivalis. Thesis. Universitas Airlangga: 
Surabaya; 2013. p. 3. 

12.  Hidayati DYN, Santosaningsih D, Khotimah H. Characterization 
of Pili protein as cell adhesion molecules of mycobacterium 
tuberculosis H37RV to mouse enterocytes: in vitro study. Int J Res 
Rev. 2018; 5(4): 66–72. 

13.  Reynolds J. Serial dilution protocols. Am Soc Microbiol. 2005; 
(September 2005): 1–7. 

14.  Ridwan RD. The role of Actinobacillus actinomycetemcomitans 
fimbrial adhesin on MMP-8 activity in aggressive periodontitis 
pathogenesis. Dent J (Majalah Kedokt Gigi). 2012; 45(4): 181–6. 

15.  Thu HM, Myat TW, Win MM, Thant KZ, Rahman S, Umeda K, 
Van Nguyen S, Icatlo FC, Higo-Moriguchi K, Taniguchi K, Tsuji T, 
Oguma K, Kim SJ, Bae HS, Choi HJ. Chicken egg yolk antibodies 
(IgY) for prophylaxis and treatment of rotavirus diarrhea in human 
and animal neonates: a concise review. Korean J Food Sci Anim 
Resour. 2017; 37(1): 1–9. 

16.  Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal 
versus polyclonal antibodies: distinguishing characteristics, 
applications, and information resources. ILAR J. 2005; 46(3): 
258–68. 

17.  Chalghoumi R, Beckers Y, Portetelle D, Théwis A. Hen egg yolk 
antibodies (IgY), production and use for passive immunization 
against bacterial enteric infections in chicken: a review. Biotechnol 
Agron Soc Env. 2009; 13(2): 295–308. 

18.  Pandit N, Changela R, Bali D, Tikoo P, Gugnani S. Porphyromonas 
gingivalis: Its virulence and vaccine. J Int Clin Dent Res Organ. 
2015; 7(1): 51. 

19.  Gani BA, Chismirina S, Hayati Z, B EW, Bachtiar BM, Wibawan 
I W T. T he abi l it y of IgY to recog n ize su r face protei ns of 
Streptococcus mutans. Dent J (Majalah Kedokt Gigi). 2009; 42(4): 
189–93. 

20.  Garrett TR, Bhakoo M, Zhang Z. Bacterial adhesion and biofilms 
on surfaces. Prog Nat Sci. 2008; 18(9): 1049–56. 

21.  Hasan A, Palmer RM. A clinical guide to periodontology: Pathology 
of periodontal disease. Vol. 216, British Dental Journal. Nature 
Publishing Group; 2014. p. 457–61.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v53.i1.p20–23

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23