Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Protective effect of an egg yolk-derived immunoglobulin (IgY) against Prevotella intermedia-mediated gingivitis Y.-Y. Hou1, Y.-H. Zhen2, D. Wang3, J. Zhu4, D.-X. Sun5, X.-T. Liu1, H.-X. Wang6, Y. Liu7, Y.-Y. Long2 and X.-H. Shu2 1 2 3 4 5 6 7
Clinical Medicine of Seven-year-program, Dalian Medical University, Dalian, China College of Pharmacy, Dalian Medical University, Dalian, China Dalian Stomatological Hospital, Dalian, China Second Affiliated Hospital of Dalian Medical University, Dalian, China First Affiliated Hospital of Dalian Medical University, Dalian, China College of Basic Medical Sciences, Dalian Medical University, Dalian, China Fifth Hospital of Wuhan, Wuhan, China
Keywords bleeding on probing, egg yolk-derived immunoglobulin, gingival index, plaque index, Prevotella intermedia, rat gingivitis. Correspondence Yu-Hong Zhen, College of Pharmacy, Dalian Medical University, No.9 Lvshun Southern Road, Lvshunkou District, Dalian 116044, China. E-mail: [email protected] Dan Wang, Dalian Stomatological Hospital, No.935 Changjiang Road, Shahekou District, Dalian 116021, China. E-mail: [email protected] 2013/1979: received 27 September 2013, revised 26 November 2013 and accepted 3 December 2013 doi:10.1111/jam.12419
Abstract Aims: To investigate the effects of an egg yolk-derived immunoglobulin (IgY) specific to Prevotella intermedia in vitro and in vivo. Methods and Results: An IgY specific to P. intermedia was produced by immunizing hens with formaldehyde-inactivated P. intermedia and showed high titres when subjected to an ELISA. The obtained IgY inhibited the growth of P. intermedia in a dose-dependent manner at concentrations from 1 to 20 mg ml 1 in Center for Disease Control and Prevention liquid medium. Forty rats were challenged with P. intermedia on gingivae and then randomly divided into four groups, which were syringed respectively with phosphatebuffered saline, 1 mg ml 1 of tinidazole, 20 mg ml 1 of nonspecific IgY and 20 mg ml 1 of the IgY specific to P. intermedia at a dosage of 300 ll per day. Gingival index (GI), plaque index (PI), bleeding on probing (BOP), counts of white blood cell (WBC) and histopathological slide of the gums were measured after treatment for 15 days. The gingivitis rats treated with the IgY specific to P. intermedia showed significantly decreased GI, PI, BOP and WBC (P < 005). Gum histopathology of the treated rats demonstrated a superior protective effect of the specific IgY on P. intermedia-mediated gingivitis. Conclusions: A new immunoglobulin specific to P. intermedia was developed from egg yolk. This specific IgY can dose-dependently inhibit the growth of P. intermedia and protect rats from gingivitis induced by P. intermedia. Significance and Impact of the Study: The new IgY has potential for the treatment of P. intermedia-mediated gingivitis.
Introduction Periodontal disease is a chronic inflammatory process that leads to the destruction of gingival connective tissue and alveolar bone and eventually causes loss of teeth (Choi et al. 2011). Recent evidence suggests that periodontal disease is a potential risk factor for several systemic diseases including cardiovascular disease, diabetes, stroke and preterm low birthweight (Choi et al. 2012). 1020
Gingivitis is one of the commonest forms of periodontal disease and one of the most widespread human infectious diseases. Although several factors are associated with gingivitis, bacterial infection is considered to be the leading cause (Signoretto et al. 2011). Gingivitis is characterized by dental plaque, which mainly composed of gram-negative strict anaerobes such as Prevotella intermedia, Porphyromonas gingivalis and Fusobacterium nucleatum. P. intermedia is one of these potential periodontopathogenic
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bacterial species (Braga et al. 2010; Estrela et al. 2010), and P. intermedia cells are frequently recovered from subgingival flora in patients with acute necrotizing ulcerative gingivitis (Chung et al. 1983) and pregnancy gingivitis (Kornman and Loesche 1980). It is generally accepted that lowering the oral bacterial biomass is an effective method for curing and preventing gingivitis. Antimicrobials, along with mechanical therapy (such as scaling and root planing), are commonly used in the treatment of periodontal disease (Xu et al. 2012), but antimicrobials are not available for some sufferers of gingivitis, such as pregnant women, the high-risk groups of gingivitis, because of the potential side effects. And the increasing prevalence of antibacterial-resistant bacteria has greatly reduced the applicability of antibacterial therapy (G€ uler et al. 2005). Therefore, alternative therapies for gingivitis are urgently needed. Egg yolk-derived immunoglobulin (IgY), which can be isolated from the egg yolks of immunized hens by several simple steps without distressing birds, is an inexpensive and easily produced antibody (Akita and Nakai 1992). IgY has attracted much attention and been recognized to be efficient in therapy and prevention of infectious diseases caused by bacteria, viruses and parasites (Carlander et al. 2000; Devi et al. 2002). Several kinds of specific IgYs have been successfully used in controlling oral disease. IgYs against Streptococcus mutans (Sentila et al. 2011) and Strep. mutans glucan-binding protein B were reported to prevent dental caries (Gani et al. 2009). Li et al. (2012) used an IgY against Solobacterium moorei to treat oral halitosis. IgYs against periodontal disease-causing P. gingivalis (Yokoyama et al. 2007a) and Fus. Nucleatum (Xu et al. 2012) were prepared and evaluated. However, the use of IgY against gingivitis-causing P. intermedia has not been reported. The objective of this study was to evaluate the in vitro and in vivo activity of an IgY specific to P. intermedia, and to assess the potential of this IgY for controlling P. intermedia-mediated gingivitis.
IgY treating P. intermedia-mediated gingivitis
1000 ml (final pH 75). There were no agar and sheep blood in the CDC liquid medium. Bacteria were cultured at 37°C in an anaerobic chamber (UniTech Bioscience, Guangzhou, China) with 85% N2, 10% H2 and 5% CO2. Immunization of hens Hens were immunized according to the reported methods (Zhen et al. 2008a). After 5 days of culture on solid medium, P. intermedia cells were removed from the plate by rinsing with sterile phosphate-buffered saline (PBS, pH 74) and diluted to 109 CFU ml 1 and then inactivated with 07% (w/v) formaldehyde for 24 h. The inactivated antigen suspensions were emulsified with an equal volume of complete and incomplete Freund’s adjuvant (Sigma-Aldrich, St Louis, MO) for the first and subsequent booster immunizations, respectively. Five Hy-Line laying hens (140 days old) housed at a commercial poultry operation were injected intramuscularly at three sites with a total volume of 15 ml for the first injection and 20 ml for two booster immunizations at 2-week interval. Eggs were collected daily from the immunized and nonimmunized hens in the same flock after the first injection and were stored at 4°C before use. Isolation and purification of IgY Egg yolks were diluted with six volumes of distilled water, and the pH was adjusted to 50. The suspension was stored at 4°C overnight and clarified by centrifugation (10 000 g). The water-soluble fraction (WSF) was collected and precipitated with 50% saturated ammonium sulphate and 14% (w/v) sodium sulphate successively. The precipitate was resuspended with distilled water and ultrafiltered using a Vivaflow 50 Tangential Flow Ultrafilter (Vivascience, Hannover, Germany) with a 100-kDa cut-off membrane and then lyophilized to obtain the purified IgY powder (Zhen et al. 2008a). SDS-PAGE
Materials and methods Bacterial strains and culture conditions Prevotella intermedia (ATCC25611) was obtained from Beijing Stomatological Hospital, Capital Medical University, Beijing, China. Bacteria were cultured on Center for Disease Control and Prevention (CDC) anaerobic blood agar or in CDC liquid medium (Ji et al. 2010). The composition of the CDC anaerobic blood agar was as follows: tryptone 15 g, soypeptone 5 g, sodium chloride 5 g, yeast extract 5 g, L-cystine 400 mg, hemin 5 mg, Vitamin K1 10 mg, agar 15 g, sheep blood 50 ml and distilled water
The purity of the IgY obtained from different phases of isolation was assessed by means of sodium dodecyl sulphate-polyacryl amide gel electrophoresis (SDS-PAGE). SDS-PAGE was conducted under nonreducing conditions with an 8% gel on a Pharmacia PhastSystem and Coomassie Brilliant Blue staining according to the manufacturer’s recommendations (Liuyi Instrument Factory, Beijing, China). IgY titre analysis The titres of the antibodies were determined by enzymelinked immunosorbent assay (ELISA) conducted on WSF
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fractions of the IgY specific to P. intermedia or nonspecific IgY (Lobbedey and Schlatterer 2003). The WSF fractions were firstly diluted 1 : 100 in PBS and then serially two-fold diluted. A microtitre plate was coated with 109 CFU ml 1 of P. intermedia. IgY (1 : 2000 dilution) from nonimmunized hens, serum (1 : 2000 dilution) from immunized hens and 09% NaCl aqueous solution were used as negative, positive and blank controls, respectively. Horseradish peroxidase-coupled rabbit antichicken IgG (Sigma-Aldrich, 1 : 30 000 dilution) and 3,3′–5,5′-tetramethylbenzidine were used as secondary antibody and substrate, respectively. The reaction was stopped by 2 mol l 1 H2SO4. All the agents were added at a volume of 100 ll per well, except for H2SO4 (50 ll per well). Optical density (OD) at 450 nm was read on microplate reader (Sunrise, Tecan, Australia) using 630 nm as a reference wavelength. While ODsample/ ODnegative ≥ 21, the maximum dilution multiple of the sample was determined as the titre of the measured IgY (Zhen et al. 2008a). Growth inhibition of the specific IgY to Prevotella intermedia The lyophilized powder of the IgY specific to P. intermedia (titre 3200) was dissolved in CDC liquid medium at concentrations of 1, 5, 10 and 20 mg ml 1, and nonspecific IgY powder was dissolved in the same medium at a concentration of 20 mg ml 1. Tinidazole at a concentration of 1 mg ml 1 was used as a positive control. P. intermedia freshly scraped from solid medium were added to the treated liquid medium at a final concentration of 106 CFU ml 1. All preparations were incubated anaerobically at 37°C for 40 h. The extent of bacterial growth was measured at 5-h interval using a Sunrise microplate reader at 600 nm. Effect of the specific IgY on rat gingivitis induced by Prevotella intermedia Fifty 6-week-old Sprague-Dawley female rats were obtained from the Animal Care Center of Dalian Medical University. Forty of the rats were fed with high-sugar diet and infected with P. intermedia as described previously (Ji et al. 2010). Briefly, two mandibular central incisors of the rats were ligatured with silk thread and inoculated with freshly prepared P. intermedia (1011 CFU ml 1). The rats were challenged with P. intermedia for six times at 2-day intervals. The infected rats were divided into four groups (n = 10) randomly and received different treatments after the last infection. The gums of the rats in the negative control group were washed with 300 ll PBS once a day. The gums of the rats in the specific 1022
IgY-treated group or the nonspecific IgY-treatment group were washed with 20 mg ml 1 of the IgY specific to P. intermedia or nonspecific IgY at a dose of 300 ll per day. The gums of rats in the positive control group were washed with 1 mg ml 1 of tinidazole at a dose of 300 ll per day. Another ten noninfected rats fed with a regular diet were set as blank control. Gingival index (GI), plaque index (PI), bleeding on probing (BOP) and white blood cell (WBC) count of the rats were measured after 15 days of treatment. The following scores were used for GI: 0 = normal gingiva; 1 = mild inflammation; 2 = moderate inflammation; 3 = severe inflammation. PI was scored from 0 to 3, with a value of 0 indicating an absence of plaque on the clinical crown and 3 indicating the presence of soft deposits covering more than two-thirds of the crown (Offenbacher et al. 2006). BOP was scored as follows: 0 = absence of bleeding; 1 = bleeding present (Nagata et al. 2008). The rats were sacrificed for histopathological examination on day 15. Light microscopy analysis of gingivae was performed on tissue slices fixed in neutral-buffered formalin, embedded in paraffin, sectioned at 5 lm and stained with haematoxylin and eosin (H and E). Stained tissue sections were examined microscopically by a scientist who did not know what treatment the experimental animals were assigned to. Animal experiments described in this article were performed according to the Experimental Animal Management Law of China and approved by the Animal Care and Ethics Committee of Dalian Medical University. Statistical analysis Statistical analysis was conducted using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL). Data were represented as means standard deviation (SD) and levels of significance were evaluated using one-way ANOVA with LSD test. The differences were considered significant at the level of P < 005. Results Isolation and purification of IgY The methods used in the study were effective for isolating and purifying IgY from egg yolks as documented by SDSPAGE. IgY has a molecular weight of 180 kDa as visualized in Fig. 1. After water dilution, salt precipitations and ultrafiltration, the purity of the IgY increased significantly. The electrophoretic patterns of the ultrafiltration fractions were in accordance with those of the standard IgY (purity >90%; Promega, Madison, WI).
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Figure 1 An 8% nonreducing SDS-PAGE of the IgY purification process. Lanes: 1, molecular weight marker; 2, water-soluble fraction; 3, fraction collected from ammonium sulphate precipitation; 4, standard IgY; 5, fraction collected from sodium sulphate precipitation; 6, fraction collected from ultrafiltration.
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Figure 3 Growth inhibitory activity of the specific IgY to Prevotella intermedia. P. intermedia (106 CFU ml 1) were cultured in Center for Disease Control and Prevention (CDC) liquid medium containing 1 mg ml 1 (●), 5 mg ml 1 (□), 10 mg ml 1 (Δ) and 20 mg ml 1 (▬) of the IgY specific to P. intermedia (titre 3200) or 20 mg ml 1 of nonspecific IgY (9) or 1 mg ml 1 of tinidazole (▲) or no IgY (■). CDC liquid medium without bacteria was used as blank control (◊). Data are presented as means SD (n = 3).
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Days after the first immunization Figure 2 Titre of the IgY specific to Prevotella intermedia. IgY (◊) produced by hens immunized with P. intermedia. IgY ( ) produced by nonimmunized hens from the same flock. IgY titre is presented by dilution times.
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Titre of the IgY specific to Prevotella intermedia ELISA revealed the ability of the new IgY binding with P. intermedia and showed the pattern of the immune response by the hens over 90 days (Fig. 2). The titre of the IgY produced by immunized hens increased after initial immunization. The highest titre (6400) was reached on day 50 and maintained from day 50 to day 70. The new IgY with high titre (≥3200) was continuously produced for more than 50 days. In contrast, the titre of the IgY produced by nonimmunized hens was extremely low. Growth inhibition of the specific IgY to Prevotella intermedia The growth of P. intermedia in liquid medium was inhibited by the IgY specific to P. intermedia in a
dose-dependent manner at concentrations ranging from 1 mg ml 1 to 20 mg ml 1 (Fig. 3). Upon addition of the IgY specific to P. intermedia in liquid medium, bacterial cells combined with the IgY and deposited at the bottom of the culture tube while the medium was clear. As the incubation progressed, with shaking, turbidity of the medium increased, indicating that bacteria were growing. The turbidity of the medium in culture with 20 mg ml 1 of the IgY specific to P. intermedia did not increase, while the negative control reached to plateau, meaning that the growth of bacteria was inhibited completely by the specific IgY. On the contrary, 20 mg ml 1 of nonspecific IgY did not show significant growth inhibition to P. intermedia. Effect of the specific IgY on rat gingivitis induced by Prevotella intermedia The gums of rats were red and swollen after challenged with P. intermedia six times, and the subgingival plaque was obvious in most rats. GI, PI and BOP of the gums in gingivitis rats decreased significantly by treating with 20 mg ml 1 of the IgY specific to P. intermedia for 15 days (P < 005) (Fig. 4). WBC level of gingivitis rats increased significantly (P < 005) compared with that of uninfected rats. After treated with the specific IgY for 15 days, the rats showed normalized WBC level (Fig. 5). Although the GI, PI, BOP and WBC levels of
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The gum lesion of gingivitis rats was further assessed by histopathological examination. The section of the negative control group showed obvious symptoms of inflammation including epithelial shedding, connective tissue proliferating and inflammatory cells infiltrating (Fig. 6b). There was no symptom of inflammation on the section of the specific IgY-treated gums (Fig. 6a). The injury degree of gums treated with nonspecific IgY (Fig. 6c) was a little weaker than that of gums without treatment, but was much more serious than that of gums treated with tinidazole (Fig. 6d) or the IgY specific to P. intermedia (Fig. 6e).
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Figure 4 Effect of the specific IgY on rat gingivitis induced by Prevotella intermedia. Rats were challenged on gums by P. intermedia and received different treatments for 15 days. The rats were treated with 20 mg ml 1 of the IgY specific to P. intermedia (titre 3200) ( ), 20 mg ml 1 of nonspecific IgY (&), 1 mg ml 1 of tinidazole (positive control) ( ) and phosphate-buffered saline (negative control) ( ). Rats not challenged with P. intermedia were used as blank control ( ). Gingival index (GI) and plaque index (PI) were graded from 0 to 3. Bleeding on probing (BOP) was scored as 0 or 1. Data are presented as means SD (n = 10). *P < 005 compared with the negative control; †P < 005 compared with the blank control.
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Figure 5 Counts of white blood cell (WBC) from gingivitis rats. Rats were bleed after 15 days of treatment with 20 mg ml 1 of the IgY specific to Prevotella intermedia (titre 3200), 20 mg ml 1 of nonspecific IgY, 1 mg ml 1 of tinidazole (positive control) or phosphate-buffered saline (negative control). Rats not challenged with P. intermedia were used as blank control. Data are presented as means SD (n = 10). *P < 005 compared with the negative control; †P < 005 compared with the blank control.
the tinidazole-treated rats also decreased significantly (P < 005) after 15 days of treatment, they were still higher than those of the specific IgY-treated rats. 1024
Prevotella intermedia are gram-negative black-pigmenting anaerobes that are aetiological agents of chronic adult periodontitis and reside as biofilms in the subgingival plaque (Wakabayashi et al. 2009). In this study, the IgY specific to P. intermedia was prepared and its effects on P. intermedia were evaluated in vitro and in vivo. In vitro study indicated the IgY specific to P. intermedia inhibited the growth of P. intermedia cells in a dosedependent manner by binding to them. The inhibitory effect of 20 mg ml 1 of this new IgY was comparable with that achieved with 1 mg ml 1 of tinidazole. Previous works have demonstrated that specific IgYs were able to inhibit the growth of gram-negative anaerobes in oral cavity, including Strep. mutans (Sentila et al. 2011), S. moorei (Li et al. 2012), P. Gingivalis (Yokoyama et al. 2007a) and Fus. nucleatum (Xu et al. 2012). The binding of IgY on bacteria may block or impair the functions of growth-related bacterial components and consequently lead to bacterial growth inhibition. Such a binding can enhance the bacterial agglutination causing a reduction in CFU rather than actual direct effects on individual bacteria (Tsubokura et al. 1997). IgYs may target the functional components of gramnegative bacteria such as their outer membrane proteins, lipopolysaccharides (LPSs), fimbriae (or pili) and flagella, which are crucial factors for bacterial colonization or bacterial pathogenicity (Li et al. 2012). LPSs from periodontal pathogens, including P. intermedia, stimulate the secretion of host inflammatory mediators, such as nitric oxide and cytokines in immune cells, and thereby initiate the host inflammatory response associated with periodontal disease (Kim et al. 2004, 2007). In our previous study, a specific IgY isolated from egg yolks of hens immunized with formaldehyde-killed Escherichia coli showed high binding activity to both E. coli and LPS in vitro (Zhen et al. 2011). Protective effects of this specific IgY against septicaemia caused by E. coli and endotoxemia induced by LPS in a mouse model had been proven (Zhen et al.
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Figure 6 Gum histopathology of Prevotella intermedia-challenged rats received different treatments for 15 days. (a) rats were not challenged (blank control); (b) infected rats were treated with phosphate-buffered saline (negative control); (c) infected rats were treated with 20 mg ml 1 of nonspecific IgY; (d) infected rats were treated with 1 mg ml 1 of tinidazole (positive control); (e) infected rats were treated with 20 mg ml 1 of the IgY specific to P. intermedia (titre 3200). Sections were stained with haematoxylin and eosin, observed with a microscope (9200).
IgY treating P. intermedia-mediated gingivitis
(a)
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2008b). The IgY was specific to both E. coli and LPS, because the antigen used to immunize hens was a mixture of E. coli and LPS released from killed E. coli. Thus, it may be deduced that the IgY specific to P. intermedia has potential to treat gingivitis or other periodontal disease mediated by P. intermedia and LPS. Successful passive immunization with IgY against dental caries has been reported in a mouse model (Xu et al. 2012), rat models (Hamada et al. 1991; Smith et al. 2001) and in human subjects (Hatta et al. 1997; Yokoyama et al. 2007b). In our study, the efficacy of the new IgY on P. intermedia-mediated gingivitis was performed on rats. The IgY specific to P. intermedia decreased the GI, PI and BOP of the gums as well as the WBC level in blood of rats with P. intermedia-mediated gingivitis. Histopathological examination also showed good protective effect of this IgY on gingivitis rats. Alveolar bone loss is a common symptom of periodontal disease. Xu et al. (2012) reported that Fus. nucleatuminfected mice showed a marked decrease in alveolar bone loss after treated with IgY specific to Fus. nucleatum. In
our study, alveolar bone loss was also observed in rats infected with P. intermedia, and the injury degree was reduced after treated with the IgY specific to P. intermedia (data not shown). Prevotella intermedia is the main cause of gingivitis, especially pregnancy gingivitis. As antibodies are isolated from yolks of immunized birds, IgYs are not harmful and especially suitable for pregnant woman, while antimicrobials and fluoride are not safe enough for treating pregnancy gingivitis (Giglio et al. 2009). The use of IgYs for passive immunization avoids bleeding animals to prepare antibodies. The production of polyclonal antibodies in eggs is convenient and economical. Egg yolk contains only a class of antibody. which can be easily isolated by precipitation techniques (Carlander et al. 2000). Averagely, 50–100 mg of IgY may be obtained from an egg, which is much higher than the amount of immunoglobulin obtained by bleeding animals (Narat 2003). In conclusion, a new IgY produced by immunizing laying hens with gingivitis-associated P. intermedia was able to inhibit the growth of P. intermedia in a dose-dependent
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manner and protect rats from gingivitis induced by P. intermedia. This specific IgY is expected to be used as a new therapeutic approach for the treatment of P. intermedia-mediated gingivitis. Acknowledgements This work was financially supported by the Outstanding Youth Foundation of Dalian, China (2009J22DW023), and Natural Science Foundation of Liaoning, China (2013023037). Conflict of interest The authors declare no conflict of interest. References Akita, E.M. and Nakai, S. (1992) Immunoglobulins from egg yolk: Isolation and purification. J Food Sci 57, 629–634. Braga, R.R., Carvalho, M.A., Brun˜a-Romero, O., Teixeira, R.E., Costa, J.E., Mendes, E.N., Farias, L.M. and Magalh~aes, P.P. (2010) Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction. Anaerobe 16, 234–239. Carlander, D., Kollberg, H., Wej aker, P. and Larsson, A. (2000) Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol Res 21, 1–6. Choi, E.Y., Jin, J.Y., Choi, J.I., Choi, I.S. and Kim, S.J. (2011) Effects of luteolin on the release of nitric oxide and interleukin-6by macrophages stimulated with Lipopolysaccharide from Prevotella intermedia. J Periodontol 82, 1509–1517. Choi, E.Y., Jin, J.Y., Lee, J.Y., Choi, J.I., Choi, I.S. and Kim, S.J. (2012) Anti-inflammatory effects and the underlying mechanisms of action of daidzein in murine macrophages stimulated with Prevotella intermedia lipopolysaccharide. J Periodontal Res 47, 204–211. Chung, C.P., Nisengard, R.J., Slots, J. and Genco, R.J. (1983) Bacterial IgG and IgM antibody titers in acute necrotizing ulcerative gingivitis. J Periodontol 54, 557–562. Devi, C.M., Bai, M.V., Lal, A.V., Umashankar, P.R. and Krishnan, L.K. (2002) An improved method for isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Methods 51, 129–138. Estrela, C.R., Pimenta, F.C., Alencar, A.H., Ruiz, L.F. and Estrela, C. (2010) Detection of selected bacterial species in intraoral sites of patients with chronic periodontitis using multiplex polymerase chain reaction. J Appl Oral Sci 18, 426–431. Gani, B.A., Chismirina, S., Hayati, Z., Winiati, B.E., Bachtiar, B.M. and Wibawan, I.W.T. (2009) The ability of IgY to
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Offenbacher, S., Lin, D., Strauss, R., McKaig, R., Irving, J., Barros, S.P., Moss, K., Barrow, D.A. et al. (2006) Effects of periodontal therapy during pregnancy on periodontal status, biologic parameters, and pregnancy outcomes: a pilot study. J Periodontol 77, 2011–2024. Sentila, R., Karthika, S., Michael, A. and Gandhimathi., (2011) Generation of egg yolk antibodies in chicken (IgY) against Streptococcus mutans and its in-vitro neutralization efficacy. Arch Appl Sci Res 3, 404–412. Signoretto, C., Marchi, A., Bertoncelli, A., Burlacchini, G., Tessarolo, F., Caola, I., Pezzati, E., Zaura, E. et al. (2011) Effects of mushroom and chicory extracts on the physiology and shape of Prevotella intermedia, a periodontopathogenic bacterium. J Biomed Biotechnol 635348, 1–8. Smith, D.J., King, W.F. and Godiska, R. (2001) Passive transfer of immunoglobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infect Immun 69, 3135– 3142. Tsubokura, K., Berndtson, E., Bogstedt, A., Kaijser, B., Kim, M., Ozeki, M. and Hammarstrom, L. (1997) Oral administration of antibodies as prophylaxis and therapy in Campylabacter jejuni-infected chickens. Clin Exp Immunol 108, 451–455. Wakabayashi, H., Yamauchi, K., Kobayashi, T., Yaeshima, T., Iwatsuki, K. and Yoshie, H. (2009) Inhibitory effects of lactoferrin on growth and biofilm formation of
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Porphyromonas gingivalis and Prevotella intermedia. Antimicrob Agents Chemother 53, 3308–3316. Xu, F.X., Xu, Y.P., Jin, L.J., Liu, H., Wang, L.H., You, J.S., Li, S.Y. and Li, X.Y. (2012) Effectiveness of egg yolk immunoglobulin (IgY) against periodontal disease-causing Fusobacterium nucleatum. J Appl Microbiol 113, 983–991. Yokoyama, K., Sugano, N., Rahman, A.K.M.S., Oshikawa, M. and Ito, K. (2007a) Activity of anti-Porphyromonas gingivalis egg yolk antibody against gingipains in vitro. Oral Microbiol Immunol 22, 352–355. Yokoyama, K., Sugano, N., Shimada, T., Shofiqur, R.A.K.M., Ibrahim, El.-S.M., Isoda, R., Umeda, K., Sa, N.V. et al. (2007b) Effects of egg yolk antibody against Porphyromonas gingivalis gingipains in periodontitis patients. J Oral Sci 49, 201–206. Zhen, Y.H., Jin, L.J., Guo, J., Li, X.Y., Lu, Y.N., Chen, J. and Xu, Y.P. (2008a) Characterization of specific egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli. Vet Microbiol 130, 126–133. Zhen, Y.H., Han, X., Guo, J., Qiu, Y., Huang, S.S., Ma, Y. and Xu, Y.P. (2008b) Protection of specific egg yolk immunoglobulin (IgY) against septicemia of mice caused by Escherichia coli. Prog Mod Biomed 8, 47–49. Zhen, Y.H., Fang, R., Ding, C., Jin, L.J., Li, X.Y., Diao, Y.P., Shu, X.H., Ma, X.C. et al. (2011) Efficacy of specific IgY for treatment of lipopolysaccharide induced endotoxemia using a mouse model. J Appl Microbiol 111, 1524–1532.
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ORIGINAL ARTICLE
Protective effect of an egg yolk-derived immunoglobulin (IgY) against Prevotella intermedia-mediated gingivitis Y.-Y. Hou1, Y.-H. Zhen2, D. Wang3, J. Zhu4, D.-X. Sun5, X.-T. Liu1, H.-X. Wang6, Y. Liu7, Y.-Y. Long2 and X.-H. Shu2 1 2 3 4 5 6 7
Clinical Medicine of Seven-year-program, Dalian Medical University, Dalian, China College of Pharmacy, Dalian Medical University, Dalian, China Dalian Stomatological Hospital, Dalian, China Second Affiliated Hospital of Dalian Medical University, Dalian, China First Affiliated Hospital of Dalian Medical University, Dalian, China College of Basic Medical Sciences, Dalian Medical University, Dalian, China Fifth Hospital of Wuhan, Wuhan, China
Keywords bleeding on probing, egg yolk-derived immunoglobulin, gingival index, plaque index, Prevotella intermedia, rat gingivitis. Correspondence Yu-Hong Zhen, College of Pharmacy, Dalian Medical University, No.9 Lvshun Southern Road, Lvshunkou District, Dalian 116044, China. E-mail: [email protected] Dan Wang, Dalian Stomatological Hospital, No.935 Changjiang Road, Shahekou District, Dalian 116021, China. E-mail: [email protected] 2013/1979: received 27 September 2013, revised 26 November 2013 and accepted 3 December 2013 doi:10.1111/jam.12419
Abstract Aims: To investigate the effects of an egg yolk-derived immunoglobulin (IgY) specific to Prevotella intermedia in vitro and in vivo. Methods and Results: An IgY specific to P. intermedia was produced by immunizing hens with formaldehyde-inactivated P. intermedia and showed high titres when subjected to an ELISA. The obtained IgY inhibited the growth of P. intermedia in a dose-dependent manner at concentrations from 1 to 20 mg ml 1 in Center for Disease Control and Prevention liquid medium. Forty rats were challenged with P. intermedia on gingivae and then randomly divided into four groups, which were syringed respectively with phosphatebuffered saline, 1 mg ml 1 of tinidazole, 20 mg ml 1 of nonspecific IgY and 20 mg ml 1 of the IgY specific to P. intermedia at a dosage of 300 ll per day. Gingival index (GI), plaque index (PI), bleeding on probing (BOP), counts of white blood cell (WBC) and histopathological slide of the gums were measured after treatment for 15 days. The gingivitis rats treated with the IgY specific to P. intermedia showed significantly decreased GI, PI, BOP and WBC (P < 005). Gum histopathology of the treated rats demonstrated a superior protective effect of the specific IgY on P. intermedia-mediated gingivitis. Conclusions: A new immunoglobulin specific to P. intermedia was developed from egg yolk. This specific IgY can dose-dependently inhibit the growth of P. intermedia and protect rats from gingivitis induced by P. intermedia. Significance and Impact of the Study: The new IgY has potential for the treatment of P. intermedia-mediated gingivitis.
Introduction Periodontal disease is a chronic inflammatory process that leads to the destruction of gingival connective tissue and alveolar bone and eventually causes loss of teeth (Choi et al. 2011). Recent evidence suggests that periodontal disease is a potential risk factor for several systemic diseases including cardiovascular disease, diabetes, stroke and preterm low birthweight (Choi et al. 2012). 1020
Gingivitis is one of the commonest forms of periodontal disease and one of the most widespread human infectious diseases. Although several factors are associated with gingivitis, bacterial infection is considered to be the leading cause (Signoretto et al. 2011). Gingivitis is characterized by dental plaque, which mainly composed of gram-negative strict anaerobes such as Prevotella intermedia, Porphyromonas gingivalis and Fusobacterium nucleatum. P. intermedia is one of these potential periodontopathogenic
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bacterial species (Braga et al. 2010; Estrela et al. 2010), and P. intermedia cells are frequently recovered from subgingival flora in patients with acute necrotizing ulcerative gingivitis (Chung et al. 1983) and pregnancy gingivitis (Kornman and Loesche 1980). It is generally accepted that lowering the oral bacterial biomass is an effective method for curing and preventing gingivitis. Antimicrobials, along with mechanical therapy (such as scaling and root planing), are commonly used in the treatment of periodontal disease (Xu et al. 2012), but antimicrobials are not available for some sufferers of gingivitis, such as pregnant women, the high-risk groups of gingivitis, because of the potential side effects. And the increasing prevalence of antibacterial-resistant bacteria has greatly reduced the applicability of antibacterial therapy (G€ uler et al. 2005). Therefore, alternative therapies for gingivitis are urgently needed. Egg yolk-derived immunoglobulin (IgY), which can be isolated from the egg yolks of immunized hens by several simple steps without distressing birds, is an inexpensive and easily produced antibody (Akita and Nakai 1992). IgY has attracted much attention and been recognized to be efficient in therapy and prevention of infectious diseases caused by bacteria, viruses and parasites (Carlander et al. 2000; Devi et al. 2002). Several kinds of specific IgYs have been successfully used in controlling oral disease. IgYs against Streptococcus mutans (Sentila et al. 2011) and Strep. mutans glucan-binding protein B were reported to prevent dental caries (Gani et al. 2009). Li et al. (2012) used an IgY against Solobacterium moorei to treat oral halitosis. IgYs against periodontal disease-causing P. gingivalis (Yokoyama et al. 2007a) and Fus. Nucleatum (Xu et al. 2012) were prepared and evaluated. However, the use of IgY against gingivitis-causing P. intermedia has not been reported. The objective of this study was to evaluate the in vitro and in vivo activity of an IgY specific to P. intermedia, and to assess the potential of this IgY for controlling P. intermedia-mediated gingivitis.
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1000 ml (final pH 75). There were no agar and sheep blood in the CDC liquid medium. Bacteria were cultured at 37°C in an anaerobic chamber (UniTech Bioscience, Guangzhou, China) with 85% N2, 10% H2 and 5% CO2. Immunization of hens Hens were immunized according to the reported methods (Zhen et al. 2008a). After 5 days of culture on solid medium, P. intermedia cells were removed from the plate by rinsing with sterile phosphate-buffered saline (PBS, pH 74) and diluted to 109 CFU ml 1 and then inactivated with 07% (w/v) formaldehyde for 24 h. The inactivated antigen suspensions were emulsified with an equal volume of complete and incomplete Freund’s adjuvant (Sigma-Aldrich, St Louis, MO) for the first and subsequent booster immunizations, respectively. Five Hy-Line laying hens (140 days old) housed at a commercial poultry operation were injected intramuscularly at three sites with a total volume of 15 ml for the first injection and 20 ml for two booster immunizations at 2-week interval. Eggs were collected daily from the immunized and nonimmunized hens in the same flock after the first injection and were stored at 4°C before use. Isolation and purification of IgY Egg yolks were diluted with six volumes of distilled water, and the pH was adjusted to 50. The suspension was stored at 4°C overnight and clarified by centrifugation (10 000 g). The water-soluble fraction (WSF) was collected and precipitated with 50% saturated ammonium sulphate and 14% (w/v) sodium sulphate successively. The precipitate was resuspended with distilled water and ultrafiltered using a Vivaflow 50 Tangential Flow Ultrafilter (Vivascience, Hannover, Germany) with a 100-kDa cut-off membrane and then lyophilized to obtain the purified IgY powder (Zhen et al. 2008a). SDS-PAGE
Materials and methods Bacterial strains and culture conditions Prevotella intermedia (ATCC25611) was obtained from Beijing Stomatological Hospital, Capital Medical University, Beijing, China. Bacteria were cultured on Center for Disease Control and Prevention (CDC) anaerobic blood agar or in CDC liquid medium (Ji et al. 2010). The composition of the CDC anaerobic blood agar was as follows: tryptone 15 g, soypeptone 5 g, sodium chloride 5 g, yeast extract 5 g, L-cystine 400 mg, hemin 5 mg, Vitamin K1 10 mg, agar 15 g, sheep blood 50 ml and distilled water
The purity of the IgY obtained from different phases of isolation was assessed by means of sodium dodecyl sulphate-polyacryl amide gel electrophoresis (SDS-PAGE). SDS-PAGE was conducted under nonreducing conditions with an 8% gel on a Pharmacia PhastSystem and Coomassie Brilliant Blue staining according to the manufacturer’s recommendations (Liuyi Instrument Factory, Beijing, China). IgY titre analysis The titres of the antibodies were determined by enzymelinked immunosorbent assay (ELISA) conducted on WSF
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fractions of the IgY specific to P. intermedia or nonspecific IgY (Lobbedey and Schlatterer 2003). The WSF fractions were firstly diluted 1 : 100 in PBS and then serially two-fold diluted. A microtitre plate was coated with 109 CFU ml 1 of P. intermedia. IgY (1 : 2000 dilution) from nonimmunized hens, serum (1 : 2000 dilution) from immunized hens and 09% NaCl aqueous solution were used as negative, positive and blank controls, respectively. Horseradish peroxidase-coupled rabbit antichicken IgG (Sigma-Aldrich, 1 : 30 000 dilution) and 3,3′–5,5′-tetramethylbenzidine were used as secondary antibody and substrate, respectively. The reaction was stopped by 2 mol l 1 H2SO4. All the agents were added at a volume of 100 ll per well, except for H2SO4 (50 ll per well). Optical density (OD) at 450 nm was read on microplate reader (Sunrise, Tecan, Australia) using 630 nm as a reference wavelength. While ODsample/ ODnegative ≥ 21, the maximum dilution multiple of the sample was determined as the titre of the measured IgY (Zhen et al. 2008a). Growth inhibition of the specific IgY to Prevotella intermedia The lyophilized powder of the IgY specific to P. intermedia (titre 3200) was dissolved in CDC liquid medium at concentrations of 1, 5, 10 and 20 mg ml 1, and nonspecific IgY powder was dissolved in the same medium at a concentration of 20 mg ml 1. Tinidazole at a concentration of 1 mg ml 1 was used as a positive control. P. intermedia freshly scraped from solid medium were added to the treated liquid medium at a final concentration of 106 CFU ml 1. All preparations were incubated anaerobically at 37°C for 40 h. The extent of bacterial growth was measured at 5-h interval using a Sunrise microplate reader at 600 nm. Effect of the specific IgY on rat gingivitis induced by Prevotella intermedia Fifty 6-week-old Sprague-Dawley female rats were obtained from the Animal Care Center of Dalian Medical University. Forty of the rats were fed with high-sugar diet and infected with P. intermedia as described previously (Ji et al. 2010). Briefly, two mandibular central incisors of the rats were ligatured with silk thread and inoculated with freshly prepared P. intermedia (1011 CFU ml 1). The rats were challenged with P. intermedia for six times at 2-day intervals. The infected rats were divided into four groups (n = 10) randomly and received different treatments after the last infection. The gums of the rats in the negative control group were washed with 300 ll PBS once a day. The gums of the rats in the specific 1022
IgY-treated group or the nonspecific IgY-treatment group were washed with 20 mg ml 1 of the IgY specific to P. intermedia or nonspecific IgY at a dose of 300 ll per day. The gums of rats in the positive control group were washed with 1 mg ml 1 of tinidazole at a dose of 300 ll per day. Another ten noninfected rats fed with a regular diet were set as blank control. Gingival index (GI), plaque index (PI), bleeding on probing (BOP) and white blood cell (WBC) count of the rats were measured after 15 days of treatment. The following scores were used for GI: 0 = normal gingiva; 1 = mild inflammation; 2 = moderate inflammation; 3 = severe inflammation. PI was scored from 0 to 3, with a value of 0 indicating an absence of plaque on the clinical crown and 3 indicating the presence of soft deposits covering more than two-thirds of the crown (Offenbacher et al. 2006). BOP was scored as follows: 0 = absence of bleeding; 1 = bleeding present (Nagata et al. 2008). The rats were sacrificed for histopathological examination on day 15. Light microscopy analysis of gingivae was performed on tissue slices fixed in neutral-buffered formalin, embedded in paraffin, sectioned at 5 lm and stained with haematoxylin and eosin (H and E). Stained tissue sections were examined microscopically by a scientist who did not know what treatment the experimental animals were assigned to. Animal experiments described in this article were performed according to the Experimental Animal Management Law of China and approved by the Animal Care and Ethics Committee of Dalian Medical University. Statistical analysis Statistical analysis was conducted using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL). Data were represented as means standard deviation (SD) and levels of significance were evaluated using one-way ANOVA with LSD test. The differences were considered significant at the level of P < 005. Results Isolation and purification of IgY The methods used in the study were effective for isolating and purifying IgY from egg yolks as documented by SDSPAGE. IgY has a molecular weight of 180 kDa as visualized in Fig. 1. After water dilution, salt precipitations and ultrafiltration, the purity of the IgY increased significantly. The electrophoretic patterns of the ultrafiltration fractions were in accordance with those of the standard IgY (purity >90%; Promega, Madison, WI).
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Figure 3 Growth inhibitory activity of the specific IgY to Prevotella intermedia. P. intermedia (106 CFU ml 1) were cultured in Center for Disease Control and Prevention (CDC) liquid medium containing 1 mg ml 1 (●), 5 mg ml 1 (□), 10 mg ml 1 (Δ) and 20 mg ml 1 (▬) of the IgY specific to P. intermedia (titre 3200) or 20 mg ml 1 of nonspecific IgY (9) or 1 mg ml 1 of tinidazole (▲) or no IgY (■). CDC liquid medium without bacteria was used as blank control (◊). Data are presented as means SD (n = 3).
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Days after the first immunization Figure 2 Titre of the IgY specific to Prevotella intermedia. IgY (◊) produced by hens immunized with P. intermedia. IgY ( ) produced by nonimmunized hens from the same flock. IgY titre is presented by dilution times.
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Titre of the IgY specific to Prevotella intermedia ELISA revealed the ability of the new IgY binding with P. intermedia and showed the pattern of the immune response by the hens over 90 days (Fig. 2). The titre of the IgY produced by immunized hens increased after initial immunization. The highest titre (6400) was reached on day 50 and maintained from day 50 to day 70. The new IgY with high titre (≥3200) was continuously produced for more than 50 days. In contrast, the titre of the IgY produced by nonimmunized hens was extremely low. Growth inhibition of the specific IgY to Prevotella intermedia The growth of P. intermedia in liquid medium was inhibited by the IgY specific to P. intermedia in a
dose-dependent manner at concentrations ranging from 1 mg ml 1 to 20 mg ml 1 (Fig. 3). Upon addition of the IgY specific to P. intermedia in liquid medium, bacterial cells combined with the IgY and deposited at the bottom of the culture tube while the medium was clear. As the incubation progressed, with shaking, turbidity of the medium increased, indicating that bacteria were growing. The turbidity of the medium in culture with 20 mg ml 1 of the IgY specific to P. intermedia did not increase, while the negative control reached to plateau, meaning that the growth of bacteria was inhibited completely by the specific IgY. On the contrary, 20 mg ml 1 of nonspecific IgY did not show significant growth inhibition to P. intermedia. Effect of the specific IgY on rat gingivitis induced by Prevotella intermedia The gums of rats were red and swollen after challenged with P. intermedia six times, and the subgingival plaque was obvious in most rats. GI, PI and BOP of the gums in gingivitis rats decreased significantly by treating with 20 mg ml 1 of the IgY specific to P. intermedia for 15 days (P < 005) (Fig. 4). WBC level of gingivitis rats increased significantly (P < 005) compared with that of uninfected rats. After treated with the specific IgY for 15 days, the rats showed normalized WBC level (Fig. 5). Although the GI, PI, BOP and WBC levels of
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The gum lesion of gingivitis rats was further assessed by histopathological examination. The section of the negative control group showed obvious symptoms of inflammation including epithelial shedding, connective tissue proliferating and inflammatory cells infiltrating (Fig. 6b). There was no symptom of inflammation on the section of the specific IgY-treated gums (Fig. 6a). The injury degree of gums treated with nonspecific IgY (Fig. 6c) was a little weaker than that of gums without treatment, but was much more serious than that of gums treated with tinidazole (Fig. 6d) or the IgY specific to P. intermedia (Fig. 6e).
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Figure 4 Effect of the specific IgY on rat gingivitis induced by Prevotella intermedia. Rats were challenged on gums by P. intermedia and received different treatments for 15 days. The rats were treated with 20 mg ml 1 of the IgY specific to P. intermedia (titre 3200) ( ), 20 mg ml 1 of nonspecific IgY (&), 1 mg ml 1 of tinidazole (positive control) ( ) and phosphate-buffered saline (negative control) ( ). Rats not challenged with P. intermedia were used as blank control ( ). Gingival index (GI) and plaque index (PI) were graded from 0 to 3. Bleeding on probing (BOP) was scored as 0 or 1. Data are presented as means SD (n = 10). *P < 005 compared with the negative control; †P < 005 compared with the blank control.
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Figure 5 Counts of white blood cell (WBC) from gingivitis rats. Rats were bleed after 15 days of treatment with 20 mg ml 1 of the IgY specific to Prevotella intermedia (titre 3200), 20 mg ml 1 of nonspecific IgY, 1 mg ml 1 of tinidazole (positive control) or phosphate-buffered saline (negative control). Rats not challenged with P. intermedia were used as blank control. Data are presented as means SD (n = 10). *P < 005 compared with the negative control; †P < 005 compared with the blank control.
the tinidazole-treated rats also decreased significantly (P < 005) after 15 days of treatment, they were still higher than those of the specific IgY-treated rats. 1024
Prevotella intermedia are gram-negative black-pigmenting anaerobes that are aetiological agents of chronic adult periodontitis and reside as biofilms in the subgingival plaque (Wakabayashi et al. 2009). In this study, the IgY specific to P. intermedia was prepared and its effects on P. intermedia were evaluated in vitro and in vivo. In vitro study indicated the IgY specific to P. intermedia inhibited the growth of P. intermedia cells in a dosedependent manner by binding to them. The inhibitory effect of 20 mg ml 1 of this new IgY was comparable with that achieved with 1 mg ml 1 of tinidazole. Previous works have demonstrated that specific IgYs were able to inhibit the growth of gram-negative anaerobes in oral cavity, including Strep. mutans (Sentila et al. 2011), S. moorei (Li et al. 2012), P. Gingivalis (Yokoyama et al. 2007a) and Fus. nucleatum (Xu et al. 2012). The binding of IgY on bacteria may block or impair the functions of growth-related bacterial components and consequently lead to bacterial growth inhibition. Such a binding can enhance the bacterial agglutination causing a reduction in CFU rather than actual direct effects on individual bacteria (Tsubokura et al. 1997). IgYs may target the functional components of gramnegative bacteria such as their outer membrane proteins, lipopolysaccharides (LPSs), fimbriae (or pili) and flagella, which are crucial factors for bacterial colonization or bacterial pathogenicity (Li et al. 2012). LPSs from periodontal pathogens, including P. intermedia, stimulate the secretion of host inflammatory mediators, such as nitric oxide and cytokines in immune cells, and thereby initiate the host inflammatory response associated with periodontal disease (Kim et al. 2004, 2007). In our previous study, a specific IgY isolated from egg yolks of hens immunized with formaldehyde-killed Escherichia coli showed high binding activity to both E. coli and LPS in vitro (Zhen et al. 2011). Protective effects of this specific IgY against septicaemia caused by E. coli and endotoxemia induced by LPS in a mouse model had been proven (Zhen et al.
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Figure 6 Gum histopathology of Prevotella intermedia-challenged rats received different treatments for 15 days. (a) rats were not challenged (blank control); (b) infected rats were treated with phosphate-buffered saline (negative control); (c) infected rats were treated with 20 mg ml 1 of nonspecific IgY; (d) infected rats were treated with 1 mg ml 1 of tinidazole (positive control); (e) infected rats were treated with 20 mg ml 1 of the IgY specific to P. intermedia (titre 3200). Sections were stained with haematoxylin and eosin, observed with a microscope (9200).
IgY treating P. intermedia-mediated gingivitis
(a)
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2008b). The IgY was specific to both E. coli and LPS, because the antigen used to immunize hens was a mixture of E. coli and LPS released from killed E. coli. Thus, it may be deduced that the IgY specific to P. intermedia has potential to treat gingivitis or other periodontal disease mediated by P. intermedia and LPS. Successful passive immunization with IgY against dental caries has been reported in a mouse model (Xu et al. 2012), rat models (Hamada et al. 1991; Smith et al. 2001) and in human subjects (Hatta et al. 1997; Yokoyama et al. 2007b). In our study, the efficacy of the new IgY on P. intermedia-mediated gingivitis was performed on rats. The IgY specific to P. intermedia decreased the GI, PI and BOP of the gums as well as the WBC level in blood of rats with P. intermedia-mediated gingivitis. Histopathological examination also showed good protective effect of this IgY on gingivitis rats. Alveolar bone loss is a common symptom of periodontal disease. Xu et al. (2012) reported that Fus. nucleatuminfected mice showed a marked decrease in alveolar bone loss after treated with IgY specific to Fus. nucleatum. In
our study, alveolar bone loss was also observed in rats infected with P. intermedia, and the injury degree was reduced after treated with the IgY specific to P. intermedia (data not shown). Prevotella intermedia is the main cause of gingivitis, especially pregnancy gingivitis. As antibodies are isolated from yolks of immunized birds, IgYs are not harmful and especially suitable for pregnant woman, while antimicrobials and fluoride are not safe enough for treating pregnancy gingivitis (Giglio et al. 2009). The use of IgYs for passive immunization avoids bleeding animals to prepare antibodies. The production of polyclonal antibodies in eggs is convenient and economical. Egg yolk contains only a class of antibody. which can be easily isolated by precipitation techniques (Carlander et al. 2000). Averagely, 50–100 mg of IgY may be obtained from an egg, which is much higher than the amount of immunoglobulin obtained by bleeding animals (Narat 2003). In conclusion, a new IgY produced by immunizing laying hens with gingivitis-associated P. intermedia was able to inhibit the growth of P. intermedia in a dose-dependent
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manner and protect rats from gingivitis induced by P. intermedia. This specific IgY is expected to be used as a new therapeutic approach for the treatment of P. intermedia-mediated gingivitis. Acknowledgements This work was financially supported by the Outstanding Youth Foundation of Dalian, China (2009J22DW023), and Natural Science Foundation of Liaoning, China (2013023037). Conflict of interest The authors declare no conflict of interest. References Akita, E.M. and Nakai, S. (1992) Immunoglobulins from egg yolk: Isolation and purification. J Food Sci 57, 629–634. Braga, R.R., Carvalho, M.A., Brun˜a-Romero, O., Teixeira, R.E., Costa, J.E., Mendes, E.N., Farias, L.M. and Magalh~aes, P.P. (2010) Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction. Anaerobe 16, 234–239. Carlander, D., Kollberg, H., Wej aker, P. and Larsson, A. (2000) Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol Res 21, 1–6. Choi, E.Y., Jin, J.Y., Choi, J.I., Choi, I.S. and Kim, S.J. (2011) Effects of luteolin on the release of nitric oxide and interleukin-6by macrophages stimulated with Lipopolysaccharide from Prevotella intermedia. J Periodontol 82, 1509–1517. Choi, E.Y., Jin, J.Y., Lee, J.Y., Choi, J.I., Choi, I.S. and Kim, S.J. (2012) Anti-inflammatory effects and the underlying mechanisms of action of daidzein in murine macrophages stimulated with Prevotella intermedia lipopolysaccharide. J Periodontal Res 47, 204–211. Chung, C.P., Nisengard, R.J., Slots, J. and Genco, R.J. (1983) Bacterial IgG and IgM antibody titers in acute necrotizing ulcerative gingivitis. J Periodontol 54, 557–562. Devi, C.M., Bai, M.V., Lal, A.V., Umashankar, P.R. and Krishnan, L.K. (2002) An improved method for isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Methods 51, 129–138. Estrela, C.R., Pimenta, F.C., Alencar, A.H., Ruiz, L.F. and Estrela, C. (2010) Detection of selected bacterial species in intraoral sites of patients with chronic periodontitis using multiplex polymerase chain reaction. J Appl Oral Sci 18, 426–431. Gani, B.A., Chismirina, S., Hayati, Z., Winiati, B.E., Bachtiar, B.M. and Wibawan, I.W.T. (2009) The ability of IgY to
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