Abstract: The growth inhibitory effect of egg yolk antibody (IgY) powder for food application was investigated on Escherichia coli O78:K80, as a model and the major cause of associated septicemic disease in broiler chickens. Laying hen’s hyperimmunized with E. coli O78:K80 bacteria for a 12 weeks period and IgY was isolated by the water-dilution method. E. coli O78:K80 was incubated with IgY (50, 100 and 150 mg mL^-1 specific and non-specific IgY powder) for 6 h during, which samples were taken at 2 h intervals. After the first immunization, the antibody specific activity in serum and egg yolk increased and became plateau on day 7 and 14, respectively. The protein concentration of serum and IgY powder did not change during the immunization period, but the ratio of gamma globulins to total serum protein and IgY purity (total IgY in protein) of IgY powder increased (p<0.05) and remained high throughout the experimental period. Specific IgY powder at the concentration of 150 mg mL^-1 decreased bacterial proliferation by 1.18 log CFU mL^-1 compared with the control group at 6 h of incubation period (p<0.01). Results of this study suggest that E. coli O78:K80 could have long term antigenic properties to induce an immune response in laying hens. The large-scale and long-term production of IgY, with high purity and high specific activity, can be attained by water dilution method. The inhibitory effect of specific IgY powder may encourage IgY powder to be applied as a feed additive to protect against E. coli O78:K80.

INTRODUCTION

Strains of Escherichia coli serotype O78 have been found to cause numerous extra- and intraintestinal clinical symptoms in various hosts (Adiri et al., 2003). Enterotoxigenic E. coli (ETEC) serotype O78, has been isolated from humans in enteritis, Newborn Meningitis (NBM) and sepsis (Babai et al., 1997; Gophna et al., 2001). In farm animals, this is one of the predominant serogroups in intestinal disease of diarrhaegenic cattle and calves (Ewers et al., 2004a) and sepsis in sheep and poultry (Ron et al., 1991). Colibacillosis is mainly a poultry disease that refers to any localized or systemic infection caused entirely or partly by E. coli (Barnes et al., 2003). This subversive disease is the primary cause of morbidity, mortality and condemnation of carcasses in the poultry industry worldwide (La Ragione and Woodward, 2002; Ewers et al., 2004b). In the past few years, both the incidence and intensity of colibacillosis have rapidly increased and may continue to become an even greater problem in the poultry industry (Altekruse et al., 2002). The infection is usually secondary to viral or mycoplasma infections or environmental stresses and most frequently involves Avian Pathogenic E. coli (APEC) of serogroups O1, O2 and O78 (Gomis et al., 2001), with the latter two constituting about 80% of the cases (Adiri et al., 2003). Vandekerchove et al. (2004) reported that E. coli strains isolated from the majority of the flocks belonged almost exclusively to the O78 serotype. Approaches to prevent and control APEC infections in the poultry industry include improved hygienic methods, vaccination, use of competitive exclusion products and finally antimicrobial chemotherapy, with increase of resistance to antimicrobial agents affecting limited success (Kwaga et al., 1994; Gomis et al., 2003; La Ragione et al., 2004; Miles et al., 2006; Knezevic and Petrovic, 2008; Lampang et al., 2008).

Egg yolk antibody (IgY), as a food grade, has been studied to substantiate the effective use for bacterial passive immunization (Kariyawasam et al., 2004; Wilkie et al., 2006). There are several benefits of using IgY as a feed additive to restrain bacterial infection. IgY can be isolated simply from egg yolk by the water-dilution method on a large-scale (Akita and Nakai, 1992, 1993) and it is relatively stable under various conditions including heat, pressure, acidity, alkalinity and proteolytic enzymes such as trypsin and chymotrypsin (Otani et al., 1991; Shimizu et al., 1992; Tini et al., 2002). The quantitative advantage of raising IgY has been presented by Gottstein and Hemmeler (1985), who showed that the amount of purified IgY produced in one month is 18 times higher than that of IgG produced in a rabbit. Therefore, large quantities and easily production of IgY with relatively high stability and compatibility with modern animal protection regulations, makes IgY as a potential practical food supplement.

Land application of poultry manure can contaminate water sources by introducing pollutants such as nutrients, organic matter, sediment, pathogens, heavy metals, hormones and antibiotics (Mishra et al., 2008). It is demonstrated that uncontrolled disposal of raw manure can potentially spread pathogens to non-target environments (Bach et al., 2002). For example, the abilities for E. coli to survive, replicate and move downwards in soil for up to two months through manure spreading has been indicated by Gagliardi and Karns (2000). Also, Kudva et al. (1998) reported the survival of E. coli O157:H7 for >1 year in hoarded non-aerated ovine manure pile that was exposed to environmental conditions. Therefore, by using IgY powder as a potential food supplement not only resistant pathogenic colonies and harmful microbial products are reduced in the intestinal content of birds, but also due to reduction in antibiotic needs in poultry production system, antibiotic residues decrease in poultry manure, soil and water. Furthermore, Kim and Patterson (2003) reported that specific IgY inhibited microbial uricase activity in broiler litter and laying hen manure leading to reduction in NH₃ generation and decreased environmental NH₃. Therefore, IgY powder as a potential antimicrobial agent may also be used as an important bio-environmental factor for protection of vital factors.

The aim of the present study was to investigate the in vitro reactivity and growth inhibitory effect of IgY powder on E. coli O78:K80 as a model. This study may result in the prospective future application of IgY powder as an effective measure for restraining the spread of enterotoxigenic E. coli infection from animals and birds to human.

MATERIALS AND METHODS

Antigen preparation: A strain of E. coli O78:K80 was obtained from Razi Vaccine and Serum Research Institute (Karaj, Iran). To ensure viability of the culture, bacteria were grown in Tryptic Soy Broth (TSB) (Merck, Darmestandt, Germany) for 6 h (until late log phase) at 37°C with shaking at 180 rpm. Cells were harvested by centrifugation at 5000x g for 15 min at 4°C (Sigma 6K15, Laboratory Centrifuges). The pellet was washed three times with phosphate-buffered saline (PBS, pH 7.2) and resuspended in PBS to a concentration of 2x10⁹ CFU mL^-1. To prepare the killed vaccine, 100 mL of the bacterial suspension was sonicated in an ice bath using a microtip (Ultraschallprozessor, Dr. Hielscher, UP200H) at maximum amplitude for 15 min and the lysate was filtered through a 0.45 μm sterile filter (Acrodisc Gelman Sciences, Mississauga, Ont.). Sterility of the filtrate was confirmed by plating it on tryptic soy agar. The vaccine was stored at -20°C, until use.

Immunization of hens: Forty, 36 weeks old Single Comb White Leghorn (SCWL) chickens were immunized intramuscularly at two different sites (0.5 mL per site) of breast muscles. Antigen was produced as described above with or without cells as the control. The bacteria were emulsified with an equal volume of Freund’s complete adjuvant for the first immunization, Freund’s incomplete adjuvant for booster immunization at 2 weeks and without adjuvant at 4 weeks after the initial immunization, as described by Ruan et al. (2005). The immunization dose was approximately 1.0x10⁹ CFU of E. coli O78:K80 in a volume of 1.0 mL equally divided between two injection sites. Eggs were collected daily for 12 weeks starting from the day of initial immunization, stored at 4°C and processed within 7 days after laying.

Total serum protein and gamma globulins concentrations: Hens were bled every week for 12 weeks starting at primary immunization for determination of serum total protein and gamma globulins concentrations. Total serum protein concentration was determined by biuret method. The share of individual protein fractions was determined by electrophoresis in tapes of gelled cellulose-acetate (Cellogel®, MALTA Chemetron, Milan, Italy). Interrelation and absolute concentration of protein fractions were determined by related software.

Isolation of egg yolk antibody (IgY): The Water-Soluble Fraction (WSF) of egg yolk was isolated as described by Akita and Nakai (1992) with minor modifications. Briefly, egg yolk was separated from egg white and the yolk membrane was punched. Yolk without membrane was transferred into a graduated cylinder and mixed with six volumes of cold acidified distilled water (pH 2.5 adjusted with 0.1 M HCl). The mixture was then adjusted to pH 5.0-5.2 and incubated at 4°C for 12 h. Following centrifugation at 12000x g and 4°C for 20 min, the supernatant was considered as WSF and stored at -20°C, until use.

IgY powder preparation: For wide food application of IgY, IgY powder as a dried, concentrated and stable form for long-term storage, may be suitable form for these intentions. To ensure that the results would not be confounded by the acidity, the WSF containing specific IgY with high activity levels and non-specific IgY as the control was neutralized with 0.1 M NaOH (adjusted to pH 7.0) and lyophilized by a freeze dryer (Freeze Dryer, DW 8030, Heto holten, Denmark) to obtain IgY powder.

Protein assay: Total protein concentration of WSF and IgY powder was determined by the Bradford method using Bovine Serum Albumin (BSA) (Sigma Chemical Co.) as the reference protein.

Enzyme-Linked Immunosorbent Assay (ELISA): Antibody specific activities in the sera and WSF were measured by ELISA as described by Sunwoo et al. (2002) with minor modification. A 96-well microtitre plate (Nunc MaxiSorp C Immunoplate) was coated overnight at 4°C with either 200 μL of 10⁶ CFUs of sonicated E. coli O78:K80 in carbonate-bicarbonate buffer (Sigma Chemical Co.) (0.05 M, pH 9.6). The plate was washed 3 times with washing solution containing 50 mM Tris buffered saline, pH 8.0 and 0.05% Tween 20 (TBS-T) (Sigma Chemical Co.). After washing, 200 μL of TBS-T was added to each well and incubated at 37°C for 30 min as a blocking step. Then, plate was washed with TBS-T and samples (100 μL) were added. Sera were diluted 1000x and the WSF, obtained by six times dilution of yolk was further diluted 167 times to obtain 1000x dilution. Diluted samples were added to the wells and incubated at 37°C for 90 min. Following incubation, the plate was washed 3 times with TBS-T and 100 μL of goat anti-chicken IgG conjugated with horseradish peroxidase (Sigma Chemical Co.), diluted 1:100000 with TBS-T, was added and incubated at 37°C for 90 min. The plate was then washed with TBS-T and incubated for 30 min with 100 μL of substrate solution (TMB/H₂O₂, 1.0:1.0) (Kirkegaard and Perry, Gaithersburg, MD). The reaction was stopped with addition of 100 μL of 2M H₂SO₄ and absorbance was measured at 450 nm using an automated spectophotometer (Ultramicroplate Reader, ELx808, Bio-Tek instruments Inc. USA). Titres were reported as the mean±standard error of the log₁₀ of the dilution in the OD of samples versus controls containing non-immunized hen’s serum or WSF.

To measure total IgY concentration of the WSF and IgY powder, ELISA experiment was performed as described above with chicken IgG ELISA quantitation kit (Bethyl Laboratories, Inc. USA), except that microtiter plate was coated with 100 μL of goat anti-chicken IgG at a final concentration of 10 μg mL^-1. Samples of WSF were diluted 1:20000 with TBS-T. Specific or non-specific IgY powder was reconstituted (10 mg mL^-1) and diluted with TBS-T (1:20000). Two-fold serial dilutions of chicken reference serum (6.25 mg mL^-1 IgG) in TBS-T (200-3.12 ng mL^-1) were used as standards to provide a relative measurement of total IgY concentration.

Growth inhibition assay: Experimental conditions utilized in our growth inhibition assays were standardized in preliminary experiments by modifications of previous reports (Lee et al., 2002a; Sunwoo et al., 2002).

The same strain of E. coli O78:K80 used as an antigen for immunizing chickens was subcultured on MacConkey agar (Merck, Darmestandt. Germany) plate at 37°C overnight and then suspended in TSB. The E. coli were grown with shaking at 180 rpm in TSB at 37°C overnight and the volume of 30% (v v^-1) of glycerol in TSB was added and stored at -80°C, until used. After subculturing of the bacteria on TSB at 37°C with shaking overnight, 0.5 mL of prepared bacterial culture were mixed with 50 mL of TSB and optical density was adjusted to 0.05 at 600 nm, corresponding to a cell density of approximately, 2.5x10⁷ CFU mL^-1. The turbidity of culture (OD at 600 nm) was measured by spectrophotometer (UNICO Spectrophotometer, 2100UV-vis USA) at 30 min intervals. The growth curve was plotted until reaching stationary phase (6 h).

Specific or non-specific IgY powder was reconstituted to 100, 200 and 300 mg mL^-1 with TSB. IgY solution was then centrifuged at 1500x g at 4°C for 20 min. The supernatant was taken and sterilized by using a 0.45-μm membrane filter. Two milliliters of specific or non-specific IgY solution were then added to the same volume of prepared E. coli O78:K80 culture and the mixtures were incubated at 37°C with shaking. Aliquots of the samples (100 μL) were taken at 0, 2, 4 and 6 h. Plate counts were performed by the Miles and Misra (1938) method on TSB agar plates in triplicate. The inoculated plates were incubated at 37°C overnight and colonies were counted to determine total number of Colony Forming Units (CFU) per mL of samples.

Statistical analysis: Bacterial growth inhibition was analyzed using the Student’s t-test. Statistical analyses of measured traits on serum, WSF and IgY powder were done using the General Linear Models (GLM) procedure of Statistical Analyses Systems (SAS, 2001). A probability level of p<0.05 and p<0.01 were considered statistically significant and high significant, respectively.

RESULTS AND DISCUSSION

Although, the antimicrobial effects of IgY against numerous microbial species have been demonstrated, however, information on using IgY powder as an effective and stable form of antibacterial agent is limited to its application against some bacterial species (Lee et al., 2002b; Sunwoo et al., 2002). Therefore, the investigation of antibacterial properties of IgY powder against specific dangerous bacterial species such as E. coli O78:K80, as an important cause of economically devastating diseases, is necessary.

Table 1:	The alterations of Total Protein (TP) and gamma Globulins (G) concentrations of serum and gamma Globulins to Total serum Protein ratio (G/TP) during the immunization period
Chickens were injected IM with sonicated E. coli O78:K80 on days 1, 14 and 28. Values are the mean of ten samples. a-eMeans with different superscript letters are significantly different (p<0.05)

Fig. 1:	The changes of antibody’s activity in serum and egg yolk (both at 1:1000 dilution: D) of immunized laying hens with E. coli O78:K80, during the immunization period. The arrows indicate the immunization times. ODi/ODc means immunized sample’s optical density to control’s optical density ratio. Values are the mean±SE of forty samples from each week

After the first immunization, the concentration of gamma globulins and gamma globulins to total serum protein ratio increased (p<0.05) and did not decrease to pre-immunization level (day 0) throughout the experimental period (Table 1). As a similar trend, the antibody specific activity in serum and egg yolk increased and became plateau on day 7 and 14, respectively (Fig. 1). Specific activity of antibodies is affected by antigen immunogenicity, which in turn is influenced by several factors, such as antigen properties, dosage, the route of administration and the adjuvant used. Surface structures of E. coli O78:K80, as a serotype of APEC, such as Lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG) and aerobactin outer membrane receptor (IutA) are capable to produce high level of antibody (Kariyawasam et al., 2004). Additionally, it has been reported that Freund’s adjuvant has the capability of keeping high levels of serum titre for a longer period when laying hens were immunized with antigen compared to using antigen without adjuvant (Sun et al., 2008). Therefore, conforming to previous reports, we assumed that E. coli O78:K80 assisting with Freund’s adjuvant could have long term antigenic properties to induce an immune response in SCWL chickens.

Serum and egg yolk specific activity remained on the highest titre, from day 7 and 14, respectively, throughout the experimental period. Sunwoo et al. (2002) suggested that the change in specific activity is not remarkable after the second booster immunization, thus the need for the second booster injection is alleviated. However, in the present research, the ratio of gamma globulins to total serum protein was significantly increased after the second booster injection (Table 1) and reached to the highest level on day 35. Therefore, it was appeared to be exigent for development of high levels of antibody activity during the immunization period. The immune specific antibody appearance in the yolk was delayed compared to that in the serum after the first immunization. This delay is likely due to the gradual accumulation of IgY during the yolk formation period by selective active transport which was postulated by Kitaguchi et al. (2008). Despite a gradual increase in the IgY specific activity in egg yolk, this fourfold increase remained consistently high for 10 weeks.

As shown in Fig. 2, after the first immunization, protein concentration in WSF did not change significantly, but total IgY concentration and IgY purity (total IgY in protein) in the WSF, with increasing of gamma globulins to total serum protein ratio, were increased about 25% (p<0.05) and remained relatively constant until the end of the experiment. The average (±Standard Deviation) concentrations of protein and total IgY and IgY purity in the nonimmunized WSF were 4.341±0.2, 1.035±0.1 mg mL^-1 of WSF and 23.84%, respectively. During the immunization period, protein and total IgY concentration of immunized WSF were 4.46±0.22 and 1.31±0.074 mg mL^-1 of WSF with 29.37% purity, indicating that 70.63% of the immunized WSF was from other livetins and lipoproteins.

Fig. 2:	Protein and total IgY concentrations of Water Soluble Fraction (WSF) obtained from non- immunized (day 0) and immunized egg yolk with E. coli O78:K80 during the experimental period. The arrows indicate the immunization time. Values are the mean±standard deviation of ten eggs in each week. *Indicates significantly lower concentration (p<0.05)

In contrast to the previous reports (Lee et al., 2002a; Sunwoo et al., 2002) suggesting total IgY concentrations in the WSF was relatively constant before and after the first immunization, the findings showed significant increase of IgY concentration in the WSF derived from serum gamma globulins. As already postulated this result indicates that all livetins of egg yolk correspond to proteins of serum, reported by Williams (1962).

Additionally, the concentration of total IgY in the immunized egg yolk was approximately, 7.86 mg mL^-1 of egg yolk. It has been indicated that the range of total IgY concentration in egg yolk from the immunized chicken is 8-25 mg mL^-1 of egg yolk (Sunwoo et al., 1996; Li et al., 1998; Sunwoo et al., 2002). In this research, the concentration of total IgY in immunized egg yolk was close to the lowest level reported. This variations reported may be due to the various methods of isolating IgY from egg yolk and measuring IgY concentration (Akita and Nakai, 1992), differences in the birds genetic lines or strains for some traits such as egg weight, egg yolk weight, the percentage of hen-day production and yolk total protein (Li et al., 1998) and finally the differences among individuals in the same strain (Carlander et al., 2001). The IgY purity of immunized WSF in the study was similar to Akita and Nakai (1993), who reported that the water-dilution method yielded IgY in the highest purity (31%) and with the similar activity compared to other methods. Results suggest that the simplicity and cost-effectiveness of water dilution method make this process as a promising way for a large-scale and long-term production of high purity IgY.

Table 2:	Characteristics of IgY powder prepared from the Water-Soluble Fraction (WSF) containing E. coli O78:K80-specific and non-specific IgY
Values are the mean of ten samples. a, bMeans with different superscript letters in each column are significantly different (p<0.05)

Protein concentration of specific and non-specific IgY powder were similar (Table 2), however, total IgY concentration and IgY purity of specific IgY powder were approximately 30% higher than the non-specific IgY powder (p<0.05). Furthermore, the purity of specific IgY powder was approximately 9% lower than that of the immunized WSF. In reconstruction step of the assay, IgY powder was not dissolved completely. Therefore, the reduction in the purity of IgY powder resulted from freeze-drying that decreased the solubility and ELISA readings of IgY, as reported by Sunwoo et al. (2002). Nevertheless, freeze-drying appears to be more appropriate for production of IgY powder, as it yields higher powder with lower moisture and higher antibody titer than spray drying (Yokoyama et al., 1992). Results of the present study postulate that the increase of gamma globulins to total serum protein ratio, after the first immunization and the reflection of this raising on WSF purity (Fig. 2) could be reason of higher specific IgY powder purity than non-specific IgY powder.

The growth curves of E. coli O78:K80, which was plotted under the same condition with the growth inhibitory assay, consisted of lag (0-2 h of incubation time), exponential (2-6 h of incubation time) and stationary phase. As shown in Fig. 3, lower levels of specific IgY powder had no inhibitory effect on bacterial growth compared to the non-specific (control) group. At the concentration of 100 mg mL^-1 of specific IgY powder, bacterial growth slightly decreased after 6 h of incubation (0.23 log CFU mL^-1). During 2-4 h of incubation time, the number of E. coli O78:K80 increased by 0.5 log CFU mL^-1 and 1.3 log CFU mL^-1 in the presence of 150 mg mL^-1 of the specific and non-specific IgY powder, respectively, indicating that bacteria in the specific treatment group proliferated 2.6 times less than the control group (p<0.01). Also, in this level, cell counts of the specific group were reduced (p<0.01) by 1.18 log CFU mL^-1 in comparison to that of the control group at 6 h of incubation time. Similarly, Sunwoo et al. (2002) reported that in the presence of 180 mg mL^-1 of specific IgY powder, the number of E. coli O157:H7 decreased significantly, but non-specific IgY powder had no inhibitory effects. Binding of specific IgY to bacterial surface components and structural alterations of the bacterial surface have been suggested as the mode of action of specific IgY to inhibit bacterial growth (Lee et al., 2002b; Sunwoo et al., 2002).

Fig. 3:	Growth inhibition curves of E. coli O78:K80 (approximately, 1x107 CFU mL-1) in the presence of the different concentrations of specific and non-specific (control: C) IgY powder (50, 100 and 150 mg mL-1). Values are the mean±SE of triplicate samples. **Indicates high significantly lower counts (p<0.01)

The findings also, suggest that the inhibitory effect of specific IgY powder was dose-dependent and the remarkable increase in the antibody specific activity of immunized WSF and a considerable increase in the total IgY concentration and IgY purity of specific IgY powder caused significant decrease in proliferation of bacteria, in comparison to the control group. Using the simple and practical immunization protocol, it is suggested that IgY could be a suitable alternative in food applications to promote antibacterial effects.

CONCLUSION

The present study used E. coli O78:K80 as a model and the major cause of associated septicemic disease in broiler chickens and it may be useful to evaluate effectiveness against a wider rang of pathogenic species. E. coli O78:K80 assisting with Freund’s adjuvant could have long term antigenic properties to induce an immune response in laying hens. The large-scale and long-term production of IgY, with high purity and high specific activity, can be attained by water dilution method as a simple, practical and economical process. Specific IgY powder at the concentration of 150 mg mL^-1 decreased bacterial proliferation significantly. This inhibitory effect may assure IgY powder to be applied as a feed additive to confer novel protection against E. coli O78:K80 in protecting humans, chickens and farm animals from gastrointestinal infection, associated septicemia and bacterial resistance to antibiotics caused by E. coli O78:K80.