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Ⅰ. INTRODUCTION

 The infections caused by bacteria such as Salmonella, Campylobacter and Escherichia coli (E. coli) lead to diarrhea in pigs. These bacterial infections result into serious problems and heavy economic losses (Botteldoorn et al., 2001). Conventionally, the diarrhea caused by E. coli can be treated with antibiotics, however, it has led to the development of resistance to antibiotics (Hariharan et al., 2004). Due to transposition of Antibiotic Resistance Genes (ARGs) between bacteria, resistance to antibiotics can impede the treatment of disease. The extensive use of antibiotics in animal production system has been banned in the whole world because of the presence of antibiotic residues in animal by-products and had possibilities of negative impact on human health on consumption of such animal by-products (Chowdhury et al., 2009). Therefore, it is necessary to discover an antibiotics alternative to prevernt or cure bacterial infections effectively, especially in pig and poultry.

 Recently, many studies have shown that antibodies from chicken egg yolk can be considered as promising alternative against some selective bacterial infections (Michael et al., 2010). Several studies have indicated that these are maternal antibodies transferred to the embryo via egg yolk (Michael et al., 2010) and are known as immunoglobulin Y (IgY), which are not similar to Immunoglobulin G (IgG) of mammals. This process of selective mechanism during evolution is known as passive immunity aiming to protect embryos and neonates from natural infectants. These evidences led to the fact that it is possible to produce antibodies from egg yolk with high efficiency and can be administered through an oral route, once there is an infection. Indeed, through trail of movement, fraction of neutralizing ability of IgY is maintained in various segments of the gastro-intestinal tract, regardless of its degradation by digestive enzymes present in the stomach and intestine. Thus, once excluding the demolishment by digestive enzyme and directly targeting to specific area in gastrointestinal tract, would be an economical method to reduce the cost of treatment. Accordingly, many reports demonstrated that oral administration of IgY can prevent and cure not only the infection of several E. coli strains such as O78:K80 (Mahdavi et al., 2010), O157:H7 (Chae et al., 2006), 987P (Sunwoo et al., 2010) in animal, but also effectively preclude the infection of other bacterium such as Helicobacter pylori, Pseudomonas aeruginosa, Salmonella enteritidis and Salmonella typhimurium (Mine and Kovacs-Nolan., 2002). Beside, these in-vivo studies have confirmed the effects of IgY specific with pathogenic antigens (Yokoyama et al., 1998; Shimamoto et al., 2002; Nilsson et al., 2008). This would open a new prospect to diagnose and effectively cure the diseases in animals and as well as humans without any risk of antibiotic residue.

 Therefore, in this study we evaluated the production of IgY against to E. coli E68 (K88) and H28 (O139:K82) antigens, which are believed as a cause of diarrhea and beriberi in piglets (Jones and Rutter, 1972; Mattsson and Wallgre, 2008). We intramuscularly injected an inactivated E68 and H28 E. coli antigen in CP-brown laying hens. The antibody titers produced in both serum and yolk were determined by Antigen-Antibody (Ag-Ab) precipitated reaction. Once succeeded, this method will enhance the specification of antibodies produced to antigen due to the distance of evolution between mammalian and poultry, minimizing the cost of production as well as unwanted reaction inducing fake results.

Ⅱ. MATERIALS AND METHODS

Chickens and Bacteria strains

 The 53-weeks-old CP-brown laying hens purchased from a local hatcher (TanUyen, BinhDuong, Vietnam) were used in this experiment. All laying hens were settled down for 2 weeks before experiment. The strain E68 E. coli induced diarrhea in piglets and O139:K82 E. coli induced edema disease in pigs were supplied from Microbiology and Infectious Disease Faculty, Department of Animal Science and Veterinary Medicine (Ho Chi Minh city University of Agriculture and Forestry, Viet Nam).

Preparation of E. coli antigens

 The preparation of E. coli antigens was done according to Orskov et al. (1977). Briefly, isolated E. coli was shaken in Trypticase Soy Broth (TSB) (BD bioscience, Heidelberg, Germany) at 37℃ overnight. Exactly 0.2 ㎖ of E. coli suspension was plated on Tryptic Soy Agar (TSA) (BD bioscience, Heidelberg, Germany) and growth 37℃ overnight. Picking up colonies was conducted by washing with 5 ㎖ Sodium Chloride (NaCl) 0.85%, followed by centrifugation 3 times at 10,000 rpm for 10 min each. Such pellet was then resuspended with 0.3% formaldehyde to inactivate E. coli. After centrifugation at 10,000 rpm for 10 minutes, the pellets were resuspended in 0.85% NaCl and concentration was adjusted as 109 CFU/㎖, and stored at 4℃.

Immunization of chickens

 Three groups of chicken (n=10 in each group) were induced immunization by injection of E68 antigen or E68 antigen with or without H28 antigen. Fifty three weeks old CP brown chickens were intramuscularly injected at three different sites (0.6 ㎖ each site), two in the breast muscles (left and right side) and one in thigh. A booster immunization was given after 10 days (0.9 ㎖ each site), 20 days (1.2 ㎖ each site) and highest dose after 30 days (0.5 ㎖ each site) of the initial immunization. Finally, eggs were collected daily and stored at 4℃ until the extraction of IgY antibody was conducted.

Purification of antibodies from egg yolk and serum

 The egg yolk was separated from white and then rolled on a paper towel to remove adhering yolk membrane. Total 30 ㎖ of yolk was diluted with 90 ㎖ of 3 mM hydrochloric acid (HCl) (ratio 1:3) and calibrated pH into 5 by adding 10% acetic acid solution. Incubation was conducted at 4℃ for 3 hours, followed by centrifuging at room temperature at 10000 rpm for 25 minutes. The supernatant was obtained and stirred with the same volume of chloroform. After incubation at 4℃ for 12 hours, supernatant was collected again by centrifugation at room temperature at 10000 rpm for 15 min. Solid sodium sulfate was gently added with final concentration 0.2 g/㎖. The precipitate was collected by centrifugation at 7000 rpm for 15 minutes and redissolved in 4 ㎖ of 10 mM Tris Buffered Saline (TBS), pH 7.3 containing 0.05% Sodium azide (NaN₃) (S2002, Louis, MO, US) and subsequently 2 ㎖ of 36% sodium sulfate was added. After centrifugation with same speed, the precipitate was dissolved in 2 ㎖ TBS (10 mM, pH 7.3) buffer containing 0.5 M sodium sulfate. Then precipitate was recovered by centrifugation, re-solubilized and desalted by dialysis to remove ammonium sulphate. Blood testing was done frequently to check the concentration of anti-E. coli antibodies in the serum. Exactly, 3 ㎖ blood was collected from wings vein and separated the serum for further analysis. After centrifugation, 1 volume of serum containing antibody was added in 0.01 volume of 0.05% NaN₃ and stored at 4℃. Each of antibodies was isolated and verified in weeks 10 to 11 after initial immunization.

Determination of antibody titer by precipitation reaction

The antibody titer was determined by Ag-Ab precipitation reaction. Soluble antigens combined with antibody in the presence of electrolyte, forming an insoluble precipitate of Ag-Ab complex. Each Ag-Ab precipitation reaction was performed in mixture with 0.6 ㎖ of antibodies either from purified eggs yolk or serum with 0.6 ㎖ antigen solution inside of test tube. NaCl 0.85% was used as negative control instead of antigen. Before results were observed, such mixture was incubated at 37℃ for 2 hours, followed by precipitating for overnight at 4℃. Antibody in egg yolk was diluted 1:2, while in serum was diluted 1:100 in the same diluent solution (0.85% NaCl).

Ⅲ. RESULTS AND DISCUSSION

The production of antibodies in chicken serum

 At the day of 12 post-injection, serum was collected and tittered. Results are shown in Table 1. In term of immune theory, antibodies are initially produced after stranger antigen infected into host body 1-2 weeks. Thus, we collected serum at second week and estimated antibodies titer. In both groups, antibody titers steadily increased from week 2 to week 5 and reached a peak at week 6 (1/110012.90 and 1/7008.14), while there was no antibody produced in negative control group. This is consistent with assumption in theory. Furthermore, with the increasing of antigens in second and third booster injections 0.9 ㎖ and 1.2 ㎖ respectively, the amount of antibodies concentration in serum was significantly augmented. A reason to interpret this phenomenon is that the memory cells in laying hens started to stimulate lymphocyte B cells due to booster injection and producing promptly a numerous of antibody. However, in group two, less of antibody presented about 1/7008.14 and approximately 64% as compared to group one. From the week 6 onward, antibodies concentration was steadily decreased, reached to 1/4405.00 at week 9 in both groups. It might be due to highest booster before 3 days of week 5 had affected on the production of antibody, beyond reach of chick tolerance and leading to the reduction afterward from week 7 to week 9. Consistently, reduction in food consumption and eggs delivered, pale in crest, loss in body weight had also observed (data not shown). Therefore, an optimization for highest booster dose should be considered once immunizing. From week 10 forward, there was a recovery with the increase of antibodies detected in group one and two. After reaching to a peak second time, 1/160013.11 in group one at week 11 and 1/13606.43 in group two at week 13, respectively. Antibodies in both groups had witnessed a reduction and lasted for 5-7 weeks later before disappeared. However, there was a difference between two groups. The amount of antibodies in group two is seem to be lesser and slowly response than those in group two. It is probably that in group two, host immune system simultaneously interacts with is a mixture of two antigens, leading to lesser specific response and causing to the lower effect and longer time in antibody production.

Table 1. Antibody titer in serum and egg yolk of laying hens (n=10)

Antibody titer from egg yolk

 A study of Patterson et al. (1962) showed that IgY is selectively transferred from laying hens serum into egg yolk through follicular epithelium (Kramer and Cho, 1970; Loeken and Roth, 1983; Al-Natour et al., 2004; Hamal et al., 2006). By an active transport mechanism, IgG from laying hens can bind with their own receptors presented on oocyte (Loeken and Roth, 1983), resulting to transport immunoglobins to offspring. The proportion of IgY is directly related with antibodies concentration in maternal serum, raising after 5-6 days post-immunization stage in serum (Szabo, 2012). It suggest that antibody in egg yolk should be present within 1 week after immunization. Herein, we conducted antibody titer at week 10 and 11 in Table 1. The results showed that antibody gained from immunized eggs ranges from 2.8%-7.3% in group I (E68) and 2-3.6% in group II (E68+H28) respectively corresponding with antibody in serum at week 9 and 10 (Fig. 1A, B). Our results were in agreement of Schade et al. (1991), those reported specific antibodies account for 2-10% of mother serum antibodies. Herein, due to less of antibodies found in case of group II, it is unavoidable that there was less of IgY. However, optimization of time after booster injection to collect effective antibodies from egg yolk is an issue should be considered. Furthermore, it is obviously that on week 9 having less of serum antibody as at week 10, represented by 1/4407.64 as compared to 1/116013.53, but in term of efficiency of antibody transferred to egg yolk, it was more effectively in one week later. This demonstrated the ratio of serum antibody is directly proportional to antibody gained in egg yolk as illustrated in Loeken and Roth (1983) and Al-Nour et al. (2004), but it might be there was a threshold in transfer. It is necessary to identify such threshold for each group and each antigen to define the right time of post-booster injection and Fig. 1. The percent efficiency of egg yolk antibody in reference to serum at week 10 (a) and week 11 (b). The data are presented as percentage of antibodies in egg yolk against antibody titer in serum considered as 100%. achieve economic effect once applying on industrial production.

Fig. 1. The percent efficiency of egg yolk antibody in reference to serum at week 10 (a) and week 11 (b). The data are presented as percentage of antibodies in egg yolk against antibody titer in serum considered as 100%.

 In conclusion, with advantages such as: quick, convenient, cost effective with less of ethical issues, production of antibody from laying hens' eggs may provide a promising alternative to replace conventional methods and may be a new approach heading to effective and green production in future.

ACKNOWLEDGMENTS

 This research was supported by the 2013 scientific programme funded by Jeju National University.