KR100280331B1 - Production of egg yolk antibody(anti-K88) against fimbrial antigen of E. coli strain K88 - Google Patents

Abstract

In the present invention, egg yolk is isolated from eggs of laying hens immunized with E. coli K88 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then thawed at room temperature and centrifuged to remove lipids and solids. The present invention relates to a method for separating egg yolk antibodies from egg yolk by filtration and lyophilization of the remaining supernatant, and by the present invention, a single antibody capable of specifically binding to E. coli K88 Fili antigen can be prepared to prevent diarrhea in piglets. In addition, it is very effective to use yolk antibodies useful for diagnosis and treatment.

Description

Separation method of egg yolk antibody against E. coli K88 phylogeny antigen {Production of egg yolk antibody (anti-K88) against fimbrial antigen of E. coli strain K88}

The present invention relates to a method for the isolation of yolk antibodies that specifically bind to diarrhea-induced E. coli K88 phylogeny antigen of livestock.

According to the swine breeding and multi-dose breeding trends, the bacterial and viral diarrheal diseases of pigs are becoming the most common and problematic diseases in pig farms. Due to the diarrhea in Esau, the mortality and growth retardation have caused economic losses to breeding farmers. In particular, diarrhea caused by Escherichia coli is known to occur in almost all breeding farms in Korea. The incidence of E. coli diarrhea is closely related to the age of newborn piglets. When diarrhea develops, there are two types of diarrhea before and after weaning. The most common causative agents of diarrhea are K88, K99, 987P, and F41, which have K antigens. These cause the diarrheal disease in almost all livestock species, although the causative bacteria differ depending on the breeder. Among them, Escherichia coli K88 is found throughout the world where pigs are raised a lot (Rapacz et al., 1986), and 10,000,000 pigs die each year, of which 50% are due to K88 ETEC (Walters et al., 1982). K88 ETEC attaches fimbriae to the small intestine, forming colony and ultimately causing diarrhea in newborn and weaning piglets (Smith et al., 1978, Payne et al., 1993b). And three serotypes of K88 fimbriae ab, ac and ad are known (Guinee et al., 1979). Many vaccines and antibiotics are used to prevent and treat diarrhea caused by E. coli K88 (Nagy et al., 1978). However, in the case of vaccines, the preventive effect is limited to newborn piglets, and antibiotics have a lot of limitations in the administration of antibiotics around the world because antibiotics are resistant to young pigs and may remain in the body when misused and abused.

Recently, studies have reported that passive immunization using maternally derived antibodies is very effective in preventing diarrhea and various diseases during neonatal period. In particular, it has been reported to prevent and treat diarrhea when oral administration of specific antibodies to toxin antigens of Escherichia coli to newborn piglets ingested with colostrum deficiency (Ozpinar et al., 1996, Yokoyama et al., 1992). Based on these results, Grand Laboratories Inc. of the United States has produced antisera preventive and therapeutic drugs against Escherichia coli and Clostridium perfringens. This antiserum agent also has the disadvantage that it is not possible to obtain the disadvantages and sufficient amount to produce a high cost and a limited time to obtain antisera, and has a problem that requires a lot of cost and technology for the separation and purification of immunoglobulin.

Accordingly, it is an object of the present invention to provide a method for isolating egg yolk antibodies that can specifically bind to the pili antigens from egg yolks that are immunized with E. coli K88 pili antigen using physical methods.

In order to achieve the above object, the present inventors separated egg yolk from eggs of laying hens immunized with Escherichia coli K88 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then melted at room temperature and centrifuged to separate lipids. And by removing the solid and the remaining supernatant was filtered and lyophilized to separate the yolk antibody from egg yolk to complete the present invention.

Hereinafter, the specific configuration and operation of the present invention.

Figure 1 is a photograph showing the results of the SDS-PAGE experiments for the separation of E. coli K88 Phili antigen.

Figure 2 is an ELISA experiment procedure to see the cross-reaction for a single antibody of various E. coli strains.

Figure 3 is a photograph showing the results of immunoblotting for the identification of E. coli K88 Fili antigen.

Figure 4 is a separation procedure of egg yolk antibody (IgY) isolated from egg yolk produced from laying hens.

Figure 5 is an ELISA experiment procedure for examining the level of antibody production for E. coli K88 Phili antigen.

6 is a graph showing changes in yolk antibody titers after immunization.

Figure 7 is an ELISA experiment procedure for assaying the specificity of the antibody produced against post-immune E. coli K88 Fili antigen.

8 is an ELISA experiment procedure for confirming the reaction between the produced E. coli K88 yolk antibody (IgY) and K88 ETEC cells.

The present invention comprises the steps of identifying and identifying a pili antigen specific to the K88 strain; It is immunized to the laying hens to produce an antibody, and the antibody is isolated and evaluated. Hereinafter, the specific configuration of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Culture of Enterocoliform Escherichia Coli K88 Strains

Enterococcal E. coli K88 was used at Animal Health Center (Veterinary Sevices Branch, Manitoba agriculture, Winnipeg, MB, Canada), and was used at 37 ° C in sterile Trypticase soy broth (BBL Microbiology Systems) to isolate fimbria antigens. After 36 hours stirring culture (150 rpm), and centrifuged (4 ℃, 3000 xg, 15min) was recovered.

Example 2: Isolation and Purification of K88 Phili Antigen

The separation and purification of K88 Philly was performed by modifying and supplementing the method of Erickson (1991). The recovered Escherichia coli was suspended in 70 ml of phosphate buffered saline solution (PBS, pH 7.2), followed by agitation for 30 minutes in water at 60 ° C. to deactivate the activity of Escherichia coli and for about 10 minutes to separate the peel from the cell membrane. Homogenization was carried out at high speed (Kinematika, GMBH, Lucerne, Switzerland). Peel and cell fragments were separated from the homogenized E. coli solution by centrifugation (14000g, 15min, 4 ℃) and membrane filtration (0.45㎛, Millipore corp, Milford, USA). Only the supernatant was collected, titrated to pH 4.0 with 2.5% citric acid and stirred at room temperature for 30 minutes. The acid treated solution was left in a refrigerator (4 ° C.) for 2 hours, and then centrifuged (14000 g, 20 min, 4 ° C.) to obtain a precipitate containing fili. The precipitate was suspended in PBS again, and the purified K88 pilus preparates were separated and recovered by repeating the aforementioned acid treatment three times. Purity of the isolated K88 fil preparations was confirmed by SDS-PAGE, and antigenicity was confirmed by immunoblotting and ELISA reactions with monoclonal antibodies.

Example 3: Qualitative Test of Escherichia Coli K88 Phili Antigen

Experimental Example 1: Protein Analysis

In order to measure the content of pili protein, Akins (1995) was performed by microwave BCA method. Assay reagents were used using a BCA protein kit (Pierce Chemical Co.) and 96-well microplates (Falcon).

The K88 Fili antigen was replaced with 2.5% citric acid in acid treatment of Erickson (1991). As a result, a protein having a protein content of about 870 µg / ml was isolated.

Experimental Example 2: SDS-PAGE

The isolated K88 peel was performed by SDS-PAGE according to the method of Laemmi (1970). The electrophoresis device was a Mini-Protein II cell Kit (Bio-Rad), and the gel was 4-20% gradient Mini-Protein. II electrophoresis apparatus (Bio-Rad Laboratories, Richmond, CA, USA) was purchased and used. Bio-Rad's Low marker and High marker were purchased and used as a marker for measuring the molecular weight of the Philly protein. Electrophoresis was performed at 200V for 50 minutes and then stained using Silver Stain Plus (Bio-Rad).

The isolated K88 pili antigen was electrophoresed with SDS-polyacrylamide gel (4-20%) and stained with Silver stain plus (Bio-Rad). As a result, the main band was shown at about 30 kDa as shown in FIG. As a result of measuring the purity of the main band, it was found that the purity was more than 98%.

Experimental Example 3: Cross Reaction Investigation by ELISA Method

K88 Fili isolated from Monoclonal Antibodies (Mab, CVL, United Kingdom) against similar enterotoxic E. coli strains (K99, K88a, K88b, 987P, F41) purchased to investigate antigenic specificity of isolated K88 Fili Investigation of the degree of cross-linking reaction with was carried out by ELISA (Enzyme linked immunosorbant assay) (Fig. 2).

Washing buffer was used by adding 0.05% Tween20 (Sigma) to 0.15M PBS (0.2M Phosphate Buffer, 0.13M NaCl, pH 7.2), the coating was used Bicarbonate buffer capsules (Sigma). As the blocking buffer, 5% skim milk (pH 7.4, Gibco) was used. As the dilution buffer, PBS-T (0.05% tween 20) and 5% skim milk were mixed in the same amount, and the substrate buffer was 0.1M sodium acetate (trihydrate, NaAc.3H ₂ O) at pH 5.5 using saturated citric acid. Titration was used. As primary antibodies, K88a, K88b, 987P, F41, and K99 monoclonal antibodies were used at a level of 0.25 μg / μl, and the secondary antibody was used as both anti-mouse IgG peroxidase conjugates (Sigma) 1,000. Diluted in fold was used, and TMB (3,3 ', 5,5'-Tetramethyl benzidine, Merck) was used as a substrate for the enzyme. The reducing agent was 3% hydrogen peroxide (Sigma) and the reaction terminator was 4N H ₂ SO ₄ . The plate was a Microtest III flexible assay plate (Falcon).

In order to examine the cross-reaction between the isolated K88 phyllin antigen and other ETEC strains (K88a, K88b, 987P, F41), monoclonal antibodies (CVL, United Kingdom), the extent of binding was determined by ELISA. Only the K88a monoclonal antibody showed a specific strong response, and it was determined to have a specificity that did not react at all with other ETEC strains (Table 1). As a result, K88, a local strain of Manitoba, Canada, was determined to have K88a specificity.

The ELISA results of cross reaction among K88 pilus protein and the monoclonal antibodies of the different E. coli strains. Fimbrial adhesin Monoclonal antibodies (CVL ^* ) K88a K88b 987P F41 K99 K88 + pilus adhesin +++ - - - -

Corporation *: Purchased from CVL (United Kingdom).

Experimental Example 4: Immunoblotting

The isolated K88 peel was electrophoresed with SDS-PAGE (12%), and then immunoblotting was performed by Gershoni and Palade (1983) using a Mini Trans-Blot Cell of Bio-Rad. Membrane was used for nitrocellulose paper (Bio-Rad, Trans-Blot Transfer Medium), and 200-fold diluted K88a monoclonal antibody (CVL, England) was used as the primary antibody. Secondary antibodies were used at 1,000-fold AP-conjugated anti-mouse IgG (Jackson, USA). NBT (5-bromo-4chloro-indolyl phospate, Sigma) and BCIP (ρ-nitro blue tetrazolium chloride, Sigma) were used as colorants.

After electrophoresis of the isolated K88 pili antigen with SDS-polyacrylamide gel (12%), the result of immunoblotting to confirm whether the band shown at about 30 kDa position is K88 pili is shown in FIG. 3. I confirmed that the band is K88 Philly.

Example 4: Production of K88 Philly Antibodies

Animals and immunization methods are as follows. Animals used for antibody production were two rabbits of New Zealand White, weighing 2.5 kg, and five laying hens from a 20-week-old White Leghorn (Shaver SX 288). 150 µg of the isolated K88 pili antigen was mixed with 0.5 ml of saline, and emulsified with 0.5 ml of Freund's complete adjuvant (FCA). The emulsion was injected into the rabbit's back and hind limbs with a total of 1 ml subcutaneous and intramuscularly injected. The laying hens were inoculated first with 4 ml of 0.25 ml each to the pectoral muscle, and boosters were inoculated 2 weeks after the first vaccination. At intervals up to 12 weeks after emulsification with Freund's incomplete adjuvant (FIA), the same procedure as the first inoculation was performed. On the other hand, blood collection and egg yolk antibody (IgY) mixture was isolated as follows. In other words, blood collection was performed weekly. Rabbits were used for experiments by collecting blood from the vein of the vein and laying hens, separating blood serum, and storing it at -20 ° C. Eggs were collected every day and stored at 8 ℃ was used for the experiment. Egg yolk antibodies were isolated weekly from egg yolk produced from laying hens. Separation of the IgY mixture was performed by modifying and supplementing the method of Akita and Nakai (1993) and Jensenius et al. (1993). The modified separation method was performed as shown in FIG. 4 based on the physical method excluding chemicals as much as possible.

That is, egg yolk was isolated from eggs of laying hens immunized with E. coli K88 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then thawed at room temperature and centrifuged to remove lipids and solids. The supernatant was filtered and lyophilized to separate the yolk antibody from the yolk.

Example 5 Purity of Antibodies (IgY) Isolated from Egg Yolk

Purity of the yolk antibody was carried out through freeze drying and spray drying depending on the physical condition and purpose of the pretreated antibody material. First, egg yolk protein fractions (eg yolk antibodies) containing IgY antibodies were separated from egg yolk based on the procedure of FIG. 2 and used for lyophilization while frozen at -20 ° C. The frozen yolk antibody was mounted on a freeze dryer adjusted to -50 ° C. to powder for 2 to 3 days. In order to measure the purity of IgY in lyophilized powder, IgY content and antibody titer before and after lyophilization were measured. IgY concentration was measured by RID (Radial Immuno-Diffusion) method, immunoassay was performed by BCA method, and antibody titer was measured by ELISA method of FIG.

Next, the powdering operation of the whole egg yolk by the spray drying method is not only egg yolk itself can be used as a rich source of nutrients for young livestock, but also by eliminating the many steps that need to be taken in FIG. This study was conducted under the hypothesis that it would have the advantage of simultaneously utilizing the effect of the antibody contained in egg yolk while minimizing the economic burden. The volume of egg yolk and the capacity of distilled water were diluted at a ratio of 2.5: 1.0 before the spray dryer, and the conditions of the spray dryer were adjusted as follows. The input temperature of the diluted yolk solution was 140 ° C., the injection temperature was 72 ° C., the dosage per hour was 5 L, and the pumping speed was 22,000 rpm. IgY antibody concentrations, antibody titers and protein concentrations before and after spray drying of egg yolk were measured to investigate the contents and titers of antibodies lost during spray drying. The experimental results are shown in Table 2.

The recovery contents of egg yolk protein, IgY and antibody titer treated by different drying methods. Items Methods of drying Freeze drying Spray drying Initial volume of Egg yolk 15 ml 2500 ml Weight of yolk powder produced 432.0 mg (100%) ^a 971 g (100%) Protein content 301.5 mg (69.8%) 205.4 g (21.2%) IgY content 158.0 mg (36.6%) 29.8 g (3.1%) Antibody iter (987P ETEC) Before treatment 260,000 (100%) b After treatment 280,000 (107.7%) 220,000 (84.6%)

A: Recovery rates of protein and IgY contents over the weight of yolk

powder

b: Percent changes of the antibody titer levels before- and after

treatment

Example 6: Antibody assay and K88 antibody specificity test

Experimental Example 5: Antibody Value Measurement

Antibody production level investigation for K88 pilus antigen was carried out by ELISA (Fig. 3). Washing buffer was used by adding 0.05% Tween 20 (sigma, P-1379) to 0.15M PBS (0.2M Phosphate Buffer, 0.13M NaCl, pH 7.2) and Bicarbonate buffer capsules (Sigma). Blocking buffer was used 5% skim milk (pH 7.4, Difco). Dilution buffer was mixed with PBS-T (0.05% tween 20) and 5% skim milk in the same amount, and substrate buffer was titrated to pH 9.8 by adding 0.5mM MgCl2 to 10% Diethanol-Amine (C4H11NO2, Sigma). Was used.

Enzyme was diluted 5,000 times with Alkaline Phosphate-conjugated AffiniPure Rabbit anti-chicken IgY (IgG) (Jackson Immuo.) And Alkaline Phosphate-conjugated AffiniPure Donkey anti-rabbit IgG (Jackson Immuno.). Phosphate substrate tablets (Phosphatase, Sigma-104) were used. 5 M NaOH was used as a reaction inhibitor. The plate was a microtest III flexible assay plate (Falcon 3991). IgY samples isolated from laying hens and rabbit antiserum (IgG) or egg yolk were used for the antibody measurement, diluted from 200 to 3 times to 145,800 times and the antibody was used for the measurement.

K88-specific antibodies in egg yolk began to produce antibodies two weeks after immunization. It started to increase sharply from 3 weeks to form the antibody value at the highest level around 5 weeks after that it showed a tendency to sharply fall to maintain a certain level (Fig. 6).

The sharp decrease after 5 weeks was lower than the level of inducing an immune response by adjusting the concentration of antigen required for immunization from about 150 µg / ml to about 100 µg / ml due to the disruption of the laboratory's antigen production plan. In addition, the immunization program, which was conducted every two weeks until two and four weeks, is extended to three weeks thereafter.

Experimental Example 6: Specificity test of the produced K88 antibody

To investigate the specificity of antibodies against K88 pilus antigen produced in post-immune rabbit serum, laying hen serum (IgG) and laying egg yolk (IgY), K99 (field), K12: K99, K88 (field), K88ab, 987P pilus antigen was used to measure the degree of reaction. The buffer used was the same as the antibody used for the measurement, the plate was used 96-well immuno-microplate (NUNC, Denmark) (Fig. 7).

20,000 folds of egg laying, anti-K88 antiserum and anti-K88 yolk antibodies produced on plates coated with 987P, K88 (field), K88ab, K99 (field) and K12: K99 phyllantigens After diluting and reacting with a secondary antibody, 5,000-fold diluted Alkaline Phosphate-conjugated AffiniPure Rabbit anti-chicken IgY (IgG) (Jackson Immuo.) And Alkaline Phosphate-conjugated AffiniPure Donkey anti-rabbit IgG (Jackson Immuno.) Were used. It was. Phosphate substrate tablets (Phosphatase, Sigma-104) were used as the substrate for the enzyme. 5 M NaOH was used as a reaction inhibitor.

As a result of the experiment, the produced serum antibody and yolk antibody responded only to the K88 and K88ab strains, and thus the specificity of the K88 antigen was confirmed as shown in Table 3.

Specificity of chicken serum, rabbit serum and yolk anti-K88 antibody against the fimbrial antigen of the different E. coli strains by ELISA. Fimbrial antigen Host antibody Rabbit serum Chicken serum Egg yolk K88 (field) (Manitoba *) K88ab (ECRC **) 987P (ECRC) K99 field (ECRC) K12: K99 (ECRC) +++++ --- +++++ --- +++++ ---

Host antibodies were diluted to 1: 20,000.

+++: 50,000 <antibody titer <100,000

500: antibody titer

Manitoba: Animal health center, Veterinary Sevices branch, Manitoba

Agricultrue, MB, Canada

ECRC: E. coli reference centre, the pensylvania state university,

USA.

Example 7 Investigation of Antigen Binding Ability of Egg Yolk Antibody (IgY)

To indirectly investigate the in vivo effects of the produced K88 yolk antibody, the degree of antigenic reaction between K88 ETEC cell and yolk antibody was tested by ELISA.

10 ml of K88 ETEC cells diluted to 2.4 × 10 9 (CFU / mL) were dispensed into sterile test tubes, and added to the IgY concentrations of 20, 30, 40, 50, 70, and 90 mg, respectively, and IgY was added. The non-group was used as a control. After the addition, shaking culture (60rpm / min) at 37 ℃, and after 2 hours 1ml each sample was heat-treated at 60 ℃ for 30 minutes and used in the experiment. Reaction degree investigation was carried out as shown (Fig. 8). The buffer used was the same as that used for the antibody measurement, and the plate was 96-well immuno-microplate (NUNC, Denmark). 2nd antibody (rabbit anti-K88 antibody; 1: 2,000) is AP-Donkey anti-rab. After mixing with IgG (Jackson, diluted in dilution buffer (1: 1,250)), it was used after incubating at 37 ℃ before 2 hours.

As shown in Table 4, the group in which the yolk antibody (IgY) 20 mg was added to 10 ml of the K88 ETEC cell prepared at the concentration of 2.4 × 109 (CFU / mL) was 109 (CFU) after 2 hours of incubation. / Ml) was 106 times (CFU / ㎖) sharply decreased by about 1000 times, and gradually increased as the concentration of IgY antibody increased. In the addition group of 30 mg or more, it was shown to decrease about 10 times more than the 20 mg (IgY) treatment group, but there is no significant difference between them.

Thus, when 20 to 50 mg of K88 yolk antibody (IgY) was added to a specimen in 1 ml, it had a binding ability to reduce K88 E. coli having a concentration of 109 (CFU / ml) to 105 to 106 (CFU / ml). It turned out.

The changes of K88 Cell concentration according to different treatment levels of IgY Treatment. Cell conc. (CFU / ml) IgY 0 mg (Control) 20 mg30 mg40 mg50 mg70 mg90 mg 2.4 × 10 ⁹ 2.3 × 10 ⁶ 5.2 × 10 ⁵ 3.0 × 10 ⁵ 1.1 × 10 ⁵ 6.9 × 10 ⁴ 8.3 × 10 ⁴

Example 8 Effect of Yolk Antibody

To evaluate the actual effect of yolk antibody, 20 pigs of 21-day-old weaning pigs were selected and artificially orally administered with K88 ETEC (1012 CFU / mL). 10 heads selected by the same method were orally administered with yolk antibody of 50 mg units of IgY each time for 2 days at 6 hour intervals after E. coli administration (antibody treatment group). All weaning pigs were treated with K88 ETEC (1012 CFU / mL) twice orally at 5 hour intervals to induce diarrhea. Feces score was measured at 2 hour intervals for 5 days and weight gain was measured daily for 2 weeks. .

The results of 3 days of diarrhea and weight gain in 21-day-old weaning pigs were administered artificially with oral administration of K88 ETEC (10 ¹² CFU / mL) and control group without oral yolk antibody. Table 5 shows.

Clinical response of 21 day-old pigs after challenge with ETEC K88 local strain and treatment with yolk antibody powder. Treatment No. of Pigs No of pigs with diarrhea on; Weightgain No. of Dead Day 0 Day 1 Day 2 (FC score) Control 10 3 (1.0) 30% 3 (2.0) 30% 4 (3.0) 40% -30.2 g 330% Ab. Treated 10 5 (1.5) 50% 0 (0.0) 0% 0 (0.0) 0% +90.0 g 00%

Diarrhea incidence after oral administration of K88 ETEC (1012 CFU / mL) was artificially administered to weaning pigs 21 days of age on the day of the test. 30% in the control group developed diarrhea and increased further thereafter to 40% after 2 days. Diarrhea also increased so much that 30% of them died at the 5th day of the test.

On the other hand, 50% of the whole day of oral administration of K88 ETEC (1012 CFU / mL) caused diarrhea, but after the confirmation of diarrhea immediately after diarrhea, all diarrhea recovered. The mean daily gain in the control group decreased by -30.2 g over the entire test period, while the antibody-administered group increased by 90.0 g.

As a result of the clinical trials, the yolk antibody was found to be effective in preventing and treating diarrhea.

Example 9: In-situ Clinical Experiment

In order to investigate the prevention and treatment effect of yolk antibody caused by K88 coliform infection, on-site clinical trials were conducted from July 1 to August 31, 1997 at H pig farm in Cheongnam-gun, Chungnam. A total of 149 newborn piglets delivered from Yorkshire x Duroc sows were selected. The control group (27 heads) was bred according to the general specification management, and the treatment group (50 heads) selected only piglets with confirmed diarrhea after birth and received yolk antibody (50 mg / dose) 3 times a day for 2 days. Oral administration was performed to determine the therapeutic effect. Two groups (72 heads) were treated with oral yolk antibody (50 mg / dose) three times every 6 hours to newborn piglets immediately after delivery to investigate the protective effect of the antibody.

As a result, three days of diarrhea and weight gain were investigated in 21-day-old weaning pig pigs after the oral administration of K88 ETEC (1012 CFU / mL) and the control group that did not receive yolk antibody and the oral administration of yolk antibody. The results are shown in Table 6.

Incidence of diarrhea in relation to the different treatment groups, in the period from birth to five days of age. Groups No. of piglets Incidence of diarrhea in the newborn piglets Recovery (%) First day of age Two-five days of age Health Diarrhea Health Diarrhea ControlTrt 1Trt 2 271659 160- 1116- 101459 172- 37.087.5100

Trt 1: Piglets were treated with egg yolk antibody (50 mg (IgY), at the

onset of diarrhea, three times a day, for two days consecutively.

Trt 2: All Piglets were treated with egg yolk antibody (50 mg (IgY),

directly after birth, three times a day, for one day.

The investigation of diarrhea incidence was carried out every morning for 5 days from the time of delivery. In each survey, the feces collected from the rectum of the piglets was diluted with 1 ml of 0.85% saline by the Swab test method, followed by centrifugation (3000 xg, 10min). ), The supernatant was collected and carried out by the measurement method established in the laboratory (Fig. 8).

Newborn piglets that did not receive yolk antibodies developed diarrhea in 11 out of 27 heads at 1 day of age, and increased at 5 days of age, with 17 heads (67%) causing diarrhea. Only 10 heads (37%) Stayed healthy. On the other hand, when yolk antibody (IgY, 50 mg) was orally administered to newborn piglets whose diarrhea was confirmed at 1 day of age, 2 days, 3 days a day, 14 of 16 heads were healed and the recovery rate was 87.5%. When yolk antibodies (IgY, 50 mg) were fed three times every six hours in every 59 piglets immediately after birth, a total of 59 piglets were prevented from diarrhea, resulting in a 100% protective effect.

Therefore, administration of yolk antibody immediately after birth has the effect of preventing 100% of E. coli diarrhea, and also has a therapeutic effect of about 87.5% even when administered to piglets in diarrhea.

As described in accordance with the above embodiments, the present invention provides an economical method for isolating egg yolk antibodies against the pili antigens unique to the K88 strain, one of E. coli strains, to provide egg yolk antibodies useful for preventing, diagnosing and treating livestock diarrhea. It is a very useful invention for the animal medicine industry because it has a very excellent effect.

Claims (1)

Egg yolk was isolated from eggs of laying hens immunized with E. coli K88 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, thawed at room temperature and centrifuged to remove lipids and solids. Separation of the yolk antibody, characterized in that to separate the yolk antibody from the yolk by filtration and lyophilization.

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