PASSIVELY ADMINISTERED ANTIBODY THAT ENHANCES FEED CONVERSION EFFICIENCY

BACTCΠRQTTND OF THE INVENTION Field of the Invention

A feed additive which increases feed conversion efficiency in animals.

Discussion of the Background

In the animal industry, antibiotics are commonly used to prevent infections, improve animal performance and treat infectious diseases. Alternative methods to replace antibiotics for improving performance have not been commercially available.

At the present time, most of the antibiotics used in animal production are different from therapeuucs used in humans. However, the emergence of multi-resistant bacteπa to any antibiotic used in animals removes the possibility ot that antibiotic ever being used as a therapeuuc agent in humans. The conunuous use of anαbiotics as preventative agents or growth promoters also causes problems with drug residues in animal produce and an increasing problem of environmental pollution. Studies have indicated that as much as 75% of the administered antibiouc may be excreted via the urine and feces back into the environment ( Addison , 1984) .

It has been demonstrated that hens injected with a foreign antigen will develop specific anttbodies and deposit them in the egg yolk. Antibodies specific to Salmonella. E- cpji, and other types of bacteria are produced by the animal when orally challenged (Ricke a aL 1988; Neighbor et al, 1991). When these antibodies are administered to the animal and it is subsequently challenged with the bacteria, the antibodies prevent infection (Bartz et al, 1980; Sherman et ai, 1983; Tacket et al, 1988; Kuhlmann et al, 1988; Yolker eial. 1988; Shmidt etal. 1989; Wiedemann et ai, 1990; Hatta etaL 1993a and 1993b; Kuroki et aL 1993). Increased growth of chicks and poults obtained from hens injected with jackbean urease was reported by Pimentel et al, 1991 and Pimentel and Cook (1988).

There are at least two possible mechanisms by which antibodies prevent infection. Antibodies have been reported to decrease the number of bacteria in the gastro-intestinal tract by preventing the bacteria from attaching to the intestinal wall. Antibodies may also decrease bacteπal numbers by binding to specific receptors on the intestinal wall or the bacteπa itself thus preventing bactenai multiplicauon flshida et ai, 1992). Either mechanism of acuon of antibodies m preventing disease would overcome the deficiencies of bacterins. In addition, antibodies do not result in the emergence of resistant strains of bacteπa or pose the threat of leaving undesirable residues in animal produce.

SUMMARY OF THE INVENTION

A method for increasing feed conversion efficiency in a mammal comprising feeding a mammal a diet containing an effective amount of an antibody which binds to urease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for increasing feed conversion efficiency in animals by augmenting their feed with antibodies which bind to urease. The preferred antigen for obtaining the antibodies is jackbean urease which is commercially available from Sigma Chemical Co. We have found that mammals and, in particular, swine show enhanced feed conversion efficiency when raised for three to four consecutive weeks on a diet containing anti-urease antibodies, at a dose of 25 to 500 mg of antibody extract per ka of feed, preferably 25 to 100 mg/ka . On the average, feed conversion in swine is enhanced 9 to 15 points, or more. Antibody extract is descπbed in Example 2. Alternauveiy, the eggs can be dried and mixed with feed without first isolating the anαbodies, as described in Example 7. Cows and sheep also grow with enhanced feed conversion if raised on feed containing anti-urease antibodies. Poulu ^* y feed containing anti-urease antibodies enhances the growth rate and feed conversion efficiency of poultry, including chickens and turkeys.

The producuon of anu-urease anubodies can be accomplished by injecting animals, such as rodents or rabbits, with urease and coilecung the blood serum which contains the anαbody. However, this procedure is costly and highly invasive to the animal.

A general method for producing antibodies which is non-invasive and more economically feasible is known. It has been observed that eggs contain 50-150 mg of various antibodies. When a hen is injected with a particular antigen, 10-20% of the antibody isolated from the eggs are specific to that antigen (Losch et al, 1986; Kuhlmann et al, 1988; Burdsail et al, 1990; Gassmann et al, 1990). The immunoglobulin class of antibodies produced in eggs has been identified as IgY which is similar to IgG produced in mammals (IgG). Egg yolk anαbodies are absorbed by piglets in a similar manner to homologous pig antibodies. Wiedemann et al (1990) observed that active antibodies were found throughout the distal jejunum in 8 week-old piglets fed lyophilized egg yolk containing antibodies. The acπvity of antibodies was improved when the animals were supplied with sodium bicarbonate which reduced the acidity in the stomach. Antibodies have been repoπed to be more resistant to degradation by gastπc acidity when they are contained in the spray-dried whole egg, as compared to puπfied spray-dried anαbodies. Yokoyama et 2l (1993) reported acαve antibodies from the stomach through the ileum two hours after the pigiets (28 day-old) were orally fed chicken anubodies. In a related study, Ishida etal (1992) demonstrated that 8 and 18 months old mice had a significantly suppressed growth of intesunal Emerobactenaceae when fed milk from nypeπmmunized cows that were injected with human gut bacteπa. Chicken anubodies can further be

protected from stomach acidity and pepsin hydrolysis by encapsulating them within liposomes (Shimuzu ei aL 1993).

Previous research on the effectiveness of chicken antibodies in preventing bacterial infections in swine has been reported. In in vitro studies, Jungling et al (1991) observed that egg yolk antibodies were effective in decreasing the adhesion of enterotoxigenic E. coji onto isolated pig enterocytes. In jn vivo tests, spray-dried egg yolk extracts containing antibodies against E. coli prevented colibacillosis in newborn piglets and calves (Yokoyama et ai, 1992 and 1993, Erhard el aL 1993) and decreased the number of days that pigs were with diarrhea ( Kellner el al, 1994).

Although chicken antibodies in general are known to protect the recipient against bacterial infections, no antibody has been shown to increase feed conversion efficiency, i.e., transforming a given amount of feed into more body weight gain. The present invention provides a method of using an antibody against an enzyme (urease) which is present in the gastro-intestinal tract to obtain increased feed utilization (feed conversion efficiency) and body weight gam in mammals.

Other features of the invenuon will become apparent in the course of the following descπpuons of exemplary embodiments which are given lor illustration of the invention and are not intended to be limiting thereof.

Example 1

A. This example illustrates the preparauon of a specific anubody against urease. At 17 weeks of age (placing hens into cages), hens were injected with 0.2 mg of urease

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type π-C (Sigma Chemical Company). The inoculum was prepared by dissolving the enzyme in 0.2 ml phosphate buffered saline (PBS; pH 7.3) and 0.2 ml complete Freund's adjuvant. The antigen preparation was injected intramuscularly into two sites, 0.2 ml in each (right and left) pectoralis muscle. A total of 0.4 ml of antigen preparation per hen was administered. A second injection was administered 5-6 week following the initial injection (about 50% egg production). In the second antigen preparation, incomplete Freund's adjuvant was used instead of complete Freund's adjuvant. Hens were re-injected with the antigen preparation every two months or when the anubody titer was determined to be low. Antibody uter was determined every four weeks by ELISA. Eggs containing the specific antibody were collected 1 week after the second injecuon. Hens were maintained in an isolated room in order to minimize outside contamination. They were under a vaccination program similar to commercial settings.

B. In another experiment the hens were injected with antigen three times, which increases the average yield of anu-urease in the eggs.

Example 2

Antibody was purified as suggested by Kwan, et al (1991). Briefly, one volume of egg yolk of Example 1 A. was mixed with 9 volumes of distilled water and left to sit overnight at 4°C. Then the aqueous poruon was centrifuged at 4000 rpm for 10 minutes, and filtered through a cheesecloth in order to remove any excess fat. Liquid was frozen for freeze drying at a future time. After freeze drying, the antibody was heat

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treated (one hour at 50°-70°C.) to improve stability. The anubody acuvity was determine by an ELISA. Before running the ELISA, protein concentrauon in the egg extract was determined by the Bradford procedure and adjusted to 1 mg/ml.

Example 3

The procedure for the ELISA was as follows:

1. Plates were coated with 100 ul urease solution (10 ug/ml) in carbonate buffer. The exact anugen concentrauon was determined with a checkerboard titraαon. The plates were incubated at 4°C overnight pnor to blocking with 1.5% bovme serum albumin for 4 hours at room temperature.

2. 100 ul of the 1 mg protein/ ml egg extract was added to each well and the plates incubated at room temperature for 30 minutes.

3. Plates were washed with PBS-Tweeπ 20 solution. 100 ul of a 1:20,000 diluαon of rabbit-anα chick IgG con _j ugated to horseradish peroxidase was added to each well. The plates were incubated at room temperature for 30 mmutes. The exact enzyme-anαbody conjugate concentrauon was determined with a checkerboard utrauon.

4. Plates were washed with the PBS-Tween and 100 ul of TMB substrate was added to each well. The plates were incubated at room temperature for 15 mmutes.

5. The reacαon was stopped with 100 ul of 2 M sulfunc acid.

6. Plates were read at 455 nm m an ELISA plate reader.

7. Titer was determine as the inverse of the last diluuon in which O.D. of the immunized egg was similar to the un-immunized control (O.D. < 100).

Example 4

This study illustrates the effect of adding purified antibody to the feed of mammals. Two experiments were conducted with starter (6-week old) piglets. Control and antibody treated feed was supplied for four weeks. Antibody dose was 50 mg/kg diet. Feed and water were supplied at libitum. Feed intake and body weight were measured weekly, feed conversion was calculated weekly and expressed as feed intake/body weight gain. The results from those two experiments are shown on tables 1 through 4. Table 1 (exp 1)

Each test group consisted of two replicates of 3 pigs each. Animals were weighed prior to the experiment and assigned to pens based on sex and body weight.

Table 2 (exp V

Table 3 (exp 2)

Table 4 (exp 2)

Example 5

An experiment was conducted with weaned pigs. Pigs were fed either a control diet or an antibody mixture for 6 weeks. Hfty mg antibody extract was mixed with 1 kilogram fine ground corn and then mixed with one metric

ton feed. Feed and water were supplied ad libitum. Body weight was measured at 2. 4 and 6 weeks. Results are shown in table 5.

Table 5.

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Example 6

This example illustrates the preparation of spray dried egg containing a urease specific antibody. Antibody-containing eggs were homogenized and

then spray dried. Antibody activity is unaffected when eggs are spray dried,

as compared to a freeze dried standard. The parameters used to spray dry are: inlet temperature/outlet temperature 140 ^β C/70°C, blower at 50% capacity, compressor at 75 % and pump at 30% capacity. All those parameters were obtained in a laboratory scale Virtis Spray Dryer. In order

to sterilize and to improve the stability of the antibody (increase shelf life),

spray dried egg was incubated at 60 ^β C for 7 days. We have run antibody samples and found that this process does not decrease the antibody activity. Since spray dried product contained fat, one gram of product was mixed

with 10 ml phosphate buffered saline (PBS) followed by 10 ml of chloroform.

That suspension was mixed thoroughly and then centrifuged at 4000 rpm

to separate the protein from the lipid fraction. The protein content of the supernatant was determined by the Bradford procedure using bovine gamma- globulin as protein standard. The extract was diluted with PBS to 1 mg protein/ml prior to antibody activity and titer determination as described in example 1 .

Example 7

Two experiments were conducted with pigs from 6 weeks of age through market weight (200 lbs body weight). The antibody this time was not isolated from the egg yolk, instead whole egg (white and yolk) was homogenized and spray dried (example 6). The amount of dried egg needed to equal the activity of the purified antibody was 250 mg/kg of diet. Results are shown in tables 6 and 7.

Animals: 30 six week old pigs (Dekalb strain), were divided into two test groups. Each test group with of 3 replicates of 5 pigs.

Table 6

Table 7

Example 8

Dried egg containing antibody was prepared as in example 4 Pigs were

maintained in the experiment for 1 6 weeks, from starter period through

market weight. The results are present in table 8.

Table 8

Example 9

This study illustrates the antibody stability on the spray dried whoie egg

when stored at room temperature and at high temperature. Spray dried egg

samples (0.5- 1 .0 gr. ) were placed in small vials (4 ml) cappeα and stored at room temperature ( 21 °C) or in an incubator at 37°C. Weekly antibody activity was measured from vials kept at those temperatures and the activity was compared to a sample that was kept at 0°C. The results are present in table 9.

Table 9

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

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