Method and product for the treatment of gastric disease

This invention describes a product obtained from the isolation and concentration of specific immunoglobulins (antibodies) derived from the mammary secretions of cows immunized with Helicobacter pylori. The product is useful in preparing formulations for the treatment and/or prevention of gastric diseases.

TECHNICAL FIELD 
This invention relates to the isolation of specific immunoglobulins which, 
in the preferred embodiment, are isolated from the colostrum or milk of 
cows immunized with Helicobacter pylori. This invention is also directed 
to a method of use of these specific antibodies in the preparation of 
novel formulations useful in the enteral treatment of gastric disease. 
BACKGROUND 
The present invention relates to a process for the production of a protein 
concentrate containing immunological factors preferably of lactic origin 
and to a specific immunoglobulin population. More particularly, this 
invention relates to active immunoglobulins exhibiting specificity against 
the microorganism, Helicobacter pylori (formerly referred to in the 
literature as Campylobacter pylori) and the use of this protein in the 
management of Helicobacter pylori colonization in the gastrointestinal 
(GI) tract. More specifically, the present invention relates to the 
injection of lactating mammals with Helicobacter pylori, subsequent 
isolation and concentration of antibodies from the colostrum (or milk) 
produced by the immunized mammals and use of this concentrate, by way of 
enteral ingestion, in reducing the infectivity of Helicobacter pylori 
resident in the gastrointestinal tract. This protein (immunoglobulin) 
concentrate is useful in the treatment of pathological sequela associated 
with Helicobacter pylori colonization of the GI tract including gastritis 
and peptic ulcer disease. 
In general, the prior art discloses the introduction and use of 
immunological factors of lactic origin into-dietetic products for newborn 
babies and infants. The oral ingestion of these dietetic products being 
intended to enable these immunological factors to be utilized in the 
development of protection against the consequences of microbial infection 
within the GI tract. 
U.S. Pat. Nos. 3,992,521 and 3,984,539 disclose a process for obtaining an 
immune product containing antibodies from the serum of a horse or cow and 
the immunoglobulin product itself. These patents do not suggest nor 
disclose the specific immunoglobulin of the present invention, nor its 
method of production and isolation, nor its intended method of use for the 
treatment of Helicobacter pylori induced gastritis. 
U.S. Pat. No. 3,123,230 discloses a method for producing antibodies which 
consists of injecting a lactating mammal with a mixture of killed 
microorganisms and isolating the antibodies from serum or milk. This 
patent does not suggest nor disclose that Helicobacter pylori can induce 
an antibody response nor the specific method of treatment employed in this 
invention. 
British Patent No. 1,573,995 discloses and claims a process for the 
production and isolation of immunoglobulins exhibiting specificity against 
Escherichia coli. This patent does not suggest that microorganisms other 
than E. coli are useful. In similar fashion, the following references 
disclose the same type of process: 1) H. Hilpert, et al., Proceedings of 
the 13th Symposium Swedish Nutritional Foundation; and 2) C. Mietens, et 
al., European J. Pediatrics, 132, 239-252, (1979). 
Ebina and colleagues disclose the immunization of cows with human rotavirus 
and the isolation of immunoglobulin to the virus from the milk of cows. 
This immunoglobulin was orally administered to children and was found to 
reduce the frequency of the outbreak of diarrhea. See Ebina, et al., The 
Lancet (Oct. 29, 1983), 1029-1030, (1983); and Ebina, et al., Med. 
Microbiol. Immunol., 174, 177-185, (1985). These references do not suggest 
nor disclose that immunoglobulins to Helicobacter pylori would be useful 
in the management Helicobacter pylori induced gastritis. 
U.S. Pat. No. 4,051,235 discloses the isolation of immunoglobulins from the 
milk of vaccinated cows by coagulating the milk, recovering the lactoserum 
(whey) and selectively precipitating the immunoglobulins with ammonium 
sulfate, followed by dialysis against water, filtration and drying. 
Seroprotection tests demonstrated that the protein concentrates of U.S. 
Pat. No. 4,051,231 provided local passive immunity in the intestine 
without resorption and without any significant loss of activity in the 
digestive tract, thereby providing generalized passive protection against 
certain enteropathogenic bacteria and/or viruses. This patent does not 
suggest nor disclose that such antibodies could serve to modify the course 
of Helicobacter pylori induced gastritis. 
Much has been published regarding Helicobacter pylori itself. Helicobacter 
pylori is approximately 0.85 um in diameter with an average length of 2.9 
um. The microorganism has a smooth coat and four to six polar flagella 
which are sheathed and have bulbous ends. In fresh cultures this organism 
appears as a slender, curved Gram-negative rod. Helicobacter pylori is 
readily distinguished from other gastric bacteria and spirochaetes by the 
absence of axial filaments in its flagella. Furthermore, optimum growth 
conditions for Helicobacter pylori are unusual and help to set it apart 
from other enteropathogens. For example, Helicobacter pylori requires a 
microaerophilic gas environment (i.e. low oxygen content) to sustain 
growth. Helicobacter pylori appears to tolerate a wide range of local pH 
conditions and is relatively resistant to acid conditions. It is believed 
that this resistance is due in part to the organism's outer protein 
structure which contains urease in large amounts resulting in the cleavage 
of urea naturally present in gastric fluid and hence, the formation of a 
buffering ammonia layer immediately around the organism. 
Although a number of spiral bacteria inhabit the mouth and lower intestinal 
tract of all mammals, what distinguishes Helicobacter pylori is the is 
observation that it is localized almost exclusively to the luminal mucosal 
surface of the stomach and duodenum and generally is found deep within the 
gastric pits. 
It is the combination of the unusual growth requirements and intestinal 
location which makes eradication and treatment of Helicobacter pylori so 
difficult. The ideal antimicrobial drug suitable for the successful 
treatment of Helicobacter pylori associated gastritis should exhibit local 
activity, be stable at low pH values and should be able to readily 
penetrate the gastric mucosa. These desirable properties of an 
antimicrobial are not easily accomplished and thus, satisfactory treatment 
of Helicobacter pylori with antimicrobials has yet to be accomplished. 
The development of an agent which is effective in the management of 
Helicobacter pylori induced gastritis would fulfill a long felt need. 
There is an emerging consensus in the field of gastroenterology that 
Helicobacter pylori is a major contributing-factor in the development of 
gastritis and peptic ulcer disease. Specifically, the following reference 
is useful in establishing the background of the present invention: 
Campylobacter pylori, E. A. J. Rauws and G. N. J. Tytgat, editors, Adis 
Press Intntl. (1989). 
In general, this reference discloses, at pages 138-139, the role of 
Helicobacter pylori in the development of gastritis and peptic ulcer 
disease. The key evidence in support of Helicobacter pylori etiology in 
these conditions is based on the observation at pages 89-103, that 
elimination of Helicobacter pylori from the stomach through the use of 
antibiotics and/or bismuth compounds leads to a remission of the gastric 
disease. 
Presently, the main therapies employed in the treatment of chronic active 
gastritis and peptic ulcer disease include the histamine H2-receptor 
antagonist's, bismuth compounds, and antibiotics. However, it is generally 
accepted that all currently used treatment modalitites are clinically 
inadequate since post-treatment relapse rates remain unacceptably high. In 
addition, several of these therapies are accompanied by significant side 
effects. For example, effective antibiotic treatment of Helicobacter 
pylori infections requires treatment over an extended duration (4-6 weeks) 
and results in the induction of diarrhea and intestinal discomfort. The 
bismuth compounds are also known to have a number of significant 
undesirable side effects. 
To date, the preferred treatment has been dominated by the use of 
H2-antagonists which result in the suppression of acid and pepsin 
secretion; however, post treatment relapse rates are extremely high. Since 
symptoniatic relief and ulcer healing are the primary aim of treatment, 
without indefinite maintenance therapy, it is becoming increasingly 
apparent that a mucosal "protective agent" having antimicrobial activity 
against Helicobacter pylori, is desirable. 
Thus, the medical community has a need for a protective agent which can be 
readily utilized in pharmaceutical and/or nutritional formulations. The 
present invention fulfills that need through the discovery that enteral 
ingestion of immunoglobulins derived from lactating mammals immunized with 
Helicobacter pylori provides such protection. 
The prior art fails to suggest, disclose or contemplate the instant 
discovery which is, in part, the use of antibodies (immunoglobulin) in the 
treatment of Helicobacter pylori infection of the gastric mucosa and to 
the antibodies themselves. 
Lactile secretion derived antibodies obtained from cows immunized with 
Helicobacter pylori will provide numerous advantages over other methods of 
immunoglobulin production. The advantages include quantity, ease and 
reproducibility of immunoglobulin isolation, ease of product preparation 
and significant cost savings as compared to antibody and product 
preparation based on other isolation methods. 
One aspect of the present invention relates to a method for producing a 
milk based product having high immunological specific activity against 
Helicobacter pylori. 
Another further aspect of this invention is the specific immunoglobulin 
itself which is produced according to the disclosed method. 
A further aspect of the present invention relates to a method for treating 
mammals in order to produce milk having immunological components which 
provide protection against Helicobacter pylori to subjects imbibing same. 
A further aspect of this invention is the use of these specific antibodies 
(immunoglobulins) in the treatment of Helicobacter pylori induced 
gastritis. 
DISCLOSURE OF THE INVENTION 
There is disclosed a composition of matter consisting of non-denatured 
immunoglobulins which exhibit specific activity to the bacterium 
Helicobacter pylori. More specifically, an immunoglobulin isolated from 
the mammary secretions of mammals exhibiting specific activity towards 
Helicobacter pylori . 
There is also disclosed a medicament for gastritis caused by Helicobacter 
pylori which comprises non-denatured immunoglobulin which exhibits 
specificity towards Helicobacter pylori. The disclosed medicament may be 
used alone or in combination with a pharmacologically and/or nutritionally 
acceptable carrier and may be in a powdered or liquid form. 
There is further disclosed a method for treating an individual suffering 
from Helicobacter pylori induced gastritis, peptic ulcer disease or other 
diseases said method consisting of administration to the individual in 
need of treatment an effective amount of a composition which contains at 
least the non-denatured immunoglobulins having specificity against 
Helicobacter pylor. 
There is also disclosed a method for producing immunoglobulins exhibiting 
specificity for Helicobacter pylori which comprises the steps of 1) 
immunizing a lactating or pregnant mammal with a cell suspension of 
Helicobacter pylori emulsified in an adjuvant; 2) obtaining the colostrum 
or milk from the mammal; and 3) isolating the immunoglobulins from the 
secretion. 
In general, the composition of matter of this invention is derived by a 
process which comprises the isolation of immunoglobulins from the mammary 
secretions of mammals immunized with Helicobacter pylori, said 
immunoglobulins exhibiting specific antimicrobial activity against 
Helicobacter pylori. 
The method for the treatment of Helicobacter pylori infections, comprises 
the oral ingestion of an effective amount of Helicobacter pylori-specific 
immunoglobulins by a patient in need of treatment, said immunoglobulins 
being derived from the mammary secretions of mammals immunized with 
Helicobacter pylori. The immunoglobulins may be ingested alone or in 
combination with other materials such as fats, oils and proteins. 
In its broadest aspect the present invention is directed to novel 
compositions which demonstrate antimicrobial activity against Helicobacter 
pylori. 
The novel composition of matter of this invention consists of the 
immunoglobulins isolated from the mammary secretions of mammals immunized 
with Helicobacter pylori which may be utilized alone or combined with 
other natural or synthetic edible products such as lipids, proteins or 
oils. Said composition of matter is readily employed alone or in 
combination with other edible products to yield admixtures which are 
useful in the treatment of gastric diseases. 
Other aspects and advantages of the invention will be apparent upon 
consideration of the following detailed description of the illustrative 
embodiments hereof. 
Best Mode 
The utility of this invention was demonstrated by the ingestion of the 
specific antibody (immunoglobulins) of this invention by Helicobacter 
pylori infected germ free piglets. Ingestion of a nutritional containing 
the specific antibody (immunoglobulins) of this invention provided the 
reduction or elimination of Helicobacter pylori induced pathology as well 
as a reduction in Helicobacter pylori bacterium colonization levels in 
various gastric epithelium regions, as determined by both agar plate 
culture reisolation and by histologic methods. 
Specific antibody to Helicobacter pylori was raised in cows (as described 
in detail below) and characterized by standard immunochemical techniques 
as described below. Additional characterization of the antibodies can be 
achieved, for example, through the assessment of their ability to 
agglutinate Helicobacter pylori, their ability to fix complement in the 
presence of the Helicobacter pylori bacteria, the ability of the 
antibodies to inhibit bacterial replication and their ability to 
specifically bind the Helicobacter pylori bacterial antigens as detected 
by common immunochemical methods such as immunofluorescence and the like. 
Following immunochemical characterization, the Helicobacter pylori specific 
antibodies were fed to Helicobacter pylori monoinfected gnotobiotic 
piglets according to the feeding regimen described below. Following 
feeding of Helicobacter pylori specific antibody, blood samples were drawn 
and the animals sacrificed for subsequent microbiological and 
histopathological assessment of the treatment protocol. The results of 
these studies were compared to Helicobacter pylori monoinfected 
gnotobiotic piglet littermates fed a nonimmune milk based on the 
nutritional product, Similac.RTM. (infant nutritional product of Ross 
Laboratories, Division of Abbott Laboratories, Columbus, Oh.) which does 
not contain specific Helicobacter pylori antibodies. 
As a result of these experiments, the inventors have discovered that 
enteral ingestion of an the immunoglobulin product containing specific 
antibodies to Helicobacter pylori results in the reduction in the levels 
of viable Helicobacter pylori contained within various regions of the 
stomach and as such provides a realistic approach for the treatment of 
Helicobacter pylori induced gastritis. The Helicobacter pylori specific 
antibodies may be employed alone (i.e. in a liquid, tablet or capsule 
form) or in combination with other pharmaceutically acceptable carriers 
such as various lipids, is proteins or oils which may al so provide 
additional nutritional and/or pharmaceutical benefits.

EXPERIMENTAL 
The following examples relate to the production and use of specific 
antibodies to Helicobacter pylori and the physiological results of such 
usage. More specifically Examples 1 and 2 relate to the production and 
characterization of the immunoglobulin material isolated from pregnant 
cows immunized with Helicobacter pylori bacteria. Table 1 sets forth the 
immunological characterization of the colostrum whey products isolated 
from cows immunized with Helicobacter pylori as compared to non-immunized 
(i.e. control) cows. This data indicates that the concentration of 
Helicobacter pylori specific antibody (immunoglobulins) levels contained 
in whey provided by Helicobacter pylori immunized cows increased by over 
one hundred fold as compared to non-immunized cows. Example 3 relates to 
the biophysical and biological characterization of the specific 
immunoglobulins isolated from immunized versus non-immunized cows and 
exhibiting activity specifically against Helicobacter pylori. Tables 2 and 
3 summarize the biological and biophysical data respectively. 
Examples 4, 5 and 6 relate to the formulation and use of this immune 
material in the feeding of gnotobiotic pigs preinfected with Helicobacter 
pylori and thus, of the utility of this material in the treatment of 
Helicobacter pylori induced gastritis. The data contained in Table 4 
indicates clearly that animals exposed to Helicobacter pylori through oral 
ingestion as described in Example 5 develop systemic (sera contained) 
antibodies to Helicobacter pylori, thereby confirming the effectiveness of 
oral treatment with Helicobacter pylori as a means of achieving 
Helicobacter pylori infection. 
Example 7 relates to the assessment of the effect of feeding this immune 
material on the levels of viable Helicobacter pylori bacteria which can 
subsequently be recovered from various gastric epithelial sites of piglets 
preinfected with Helicobacter pylori. Tables 5 and 6 summarize these 
results. The data indicate markedly reduced recoveries of viable 
Helicobacter pylori from all gastric regions examined for animals fed the 
immune product as compared to animals which received the nonimmune 
nutrient only. These results clearly indicate the effectiveness of using 
Helicobacter pylori specific antibodies (immunoglobulins) in the treatment 
of Helicobacter pylori induced gastritis. 
BEST MODE FOR CARRYING OUT THE INVENTION 
EXAMPLE 1 
Preparation of Antibodies and Control Products 
Whey containing Helicobacter pylori specific antibodies was prepared from 
colostrum derived from a cow immunized while pregnant with whole formalin 
killed Helicobacter pylori bacteria (ATTCC Strain 26695). The bacteria, 
emulsified in incomplete Freund's adjuvant, were employed at a 
concentration of 5.times.10.sup.9 colony forming units (CFU)/mL. Each 
innoculation consisted of 12 mL of this material. The following 
immunization schedule was employed for each cow. Initially a subcutaneous 
(SQ) innoculation 14 days prior to drying off (D-14) was given. This was 
followed by an intramammary booster given seven days post drying off (D+7) 
and a second SQ booster given at D+30. This and similar immunization 
schedules are taught by the prior art and while the above schedule fully 
describes the method used, this description is not meant to limit the 
method of immunization under which the antibodies (immunoglobulins) to 
Helicobacter pylori can be raised since those skilled in the art will 
recognize and understand that other immunization methods would give 
similar results. 
Upon the birth of the calf of the immunized cow, colostrum was collected, 
rennet whey prepared and the whey stored frozen at -20.degree. C. until 
use. The isolation scheme for obtaining the whey is largely as described 
in U.S. Pat. No. 4,051,235 which is herein incorporated by reference. The 
basic steps involved are: 
Collection and freezing of the bovine colostrum. Thereafter fat is removed 
from the colostrum. This is achieved by partially thawing the frozen 
colostrum and removing the upper liquid portion. The remaining material is 
then completely thawed and centrifugally separated to remove as much of 
the remaining fat as possible. 
The defatted colostrum was precipitated by adding 1.5 mg of calcium 
chloride per liter of colostrum and by adding 1.5 gram of commercially 
available rennin per liter of colostrum. The mixture was then thoroughly 
stirred at 20.degree. C. Thereafter, the solution was permitted to stand 
for 2-5 hours while the casein in the solution precipitated. The 
precipitated casein was then removed by filtration. The resulting solution 
is hereinafter referred to as "bovine colostrum whey" (BCW). The solution 
was then clarified by filtration and the protein and immunoglobulin 
concentration were then determined using methods described below. 
EXAMPLE 2 
Characterization of Antibody Test Material 
The bovine colostrum whey (BCW) samples were analyzed for total protein, 
immunoglobulin isotype type characterization, IgGl content and for 
specific anti-Helicobacter pylori antibodies. The techniques used for 
these assays are standard procedures employed in the art and were, 
respectively, dye binding methods (BioRad), radial immunodiffusion, (ICN 
Biochemicals), and the enzyme linked immunoassay (ELISA). 
The ELISA plates were coated with a Helicobacter pylori cell lysate at 3.2 
ug/ml. The detecting antibody employed was a conjugate of alkaline 
phosphatase-coupled to a monoclonal antibody having immunospecificity for 
bovine IgGl type antibodies. The indicator substrate for the assay was 
p-nitrophenylphosphate. The extent of color development was measured on a 
Dynatech ELISA plate reader at a visible wavelength of 490 nm and the data 
analyzed according to standard statistical methods. The results for these 
studies are summarized in Table 1. The data illustrates clearly that oral 
exposure to Helicobacter pylori results in over a one hundred fold 
increase in immunoglobulin (IgGl) concentration in colostral whey as 
compared to whey obtained from non-immunized cows. 
TABLE 1 
__________________________________________________________________________ 
CHARACTERIZATION OF BOVINE COLOSTRAL WHEY PREATION 
ELISA.sup.b 
COLOSTRAL WHEY 
TOTAL PROTEIN 
mg IgGl/ FOLD DIFFERENCE 
PREATION.sup.a 
(mg/mL) mL TITER/mg IgGl.sup.b 
FROM NON-IMMUNE 
__________________________________________________________________________ 
IMMUNE 176.2 125.2 
615 123 
CONTROL Not Determined 
171.2 
5 1 
(Non-Immunized) 
__________________________________________________________________________ 
.sup.a Colostral whey obtained from Helicobacter pylori immunized cow and 
from the nonHelicobacter pylori immunized cow (CONTROL). 
.sup.b Nonadjusted ELISA titers on undiluted colostral whey [IMMUNE = 
77,000 and CONTROL = 860]. This assay detects antibodies specifically 
against Helicobacter pylori. 
EXAMPLE 3 
Characterization of Helicobacter pylori Antibodies 
Specific Helicobacter pylori antibodies may be characterized in a number of 
ways. For the purpose of the present study the biological activity of the 
immunoglobulins was determined by means of an agglutination assay (Table 
2) and the biophysical characterization is provided by a consideration of 
the physical properties of the dominant immunoglobulin isotype contained 
in BCW (Table 3). 
The bacterial agglutination test is a classical procedure used to determine 
the presence and relative concentration of specific antibody in sera to 
specific bacteria (e.g. Widal test for typhoid fever). Agglutination 
involves the aggregation of bacteria into large clumps as a result of the 
binding of specific antibody to particular sites on the outer surface 
antigens of the bacterium. Thus, bacterial clumping (agglutination) can be 
readily observed following the mixing of a suitable bacterial suspension 
and an immunoglobulin preparation from an animal previously immunized with 
that bacterium. Estimation of the strength (titer) of a given antibody 
preparation is accomplished by diluting the antibody (e.g. 2-fold serial 
dilutions) and determining the last dilution which shows an agglutination 
effect. Visualization of bacterial agglutination is made by examining the 
pattern of sedimented bacteria on the bottom of a U-shaped plastic 
microtiter plate. Non-agglutinated bacteria sediment into a tight button 
whereas agglutinated bacteria are hindered from forming a button and 
sediment into a diffuse pattern on the bottom of the plastic well. 
Test antibody dilutions were made in microtiter plates followed by the 
addition of appropriately diluted bacteria (formalin fixed Helicobacter 
pylori or E Coli). Buffer containing methylene blue was then added to 
facilitate easy reading of agglutination end points. The plates were 
examined after an eighteen (18) hour incubation at 4.degree. C. 
Thereafter, the extent of agglutination was determined and the results 
expressed as the lowest serial dilution of antibody which exhibited 
agglutination as defined above. 
The results of this study are shown in Table 2. The data indicate clearly 
that antibodies raised against Helicobacter pylori and contained within 
BCW react specifically at high titers (i.e. dilutions) to Helicobacter 
pylori and do not react significantly with other viral and bacterial 
antigens. Likewise, similar immunoglobulin preparations from non-immunized 
is cows or cows immunized with unrelated bacterial or viral antigens do 
not react significantly with Helicobacter pylori. 
TABLE 2 
__________________________________________________________________________ 
CHARACTERIZATION OF HELICOBACTER PYLORI (H. pylori) IMMUNE AND NON- 
IMMUNE BOVINE LACTOIMMUNOGLOBULIN PREATIONS BY BACTERIAL 
AGGLUTINATION.sup.a 
ANTIBODY TEST SAMPLE AGGLUTINATING ANTIBODY 
COLOSTRUM 
IMMUNIZING 
IgGl TEST SPECIFIC ACTIVITY 
REFERENCE 
ANTIGEN (mg/mL) 
ANTIGEN 
TITER/mL 
(TITER/mgIgGl) 
__________________________________________________________________________ 
1 H. pylori 
75.4 H. pylori 
256,000 
3,400 
2 HRV.sup.b 
52.4 H. pylori 
2,560 49 
3 E. coli 10 H. pylori 
320 32 
4 None 27.3 H. pylori 
1,280 47 
1 H. pylori 
75.4 E. coli 
640 8.5 
2 HRV 52.4 E. coli 
640 12 
3 E. coli 10 E. coli 
8,000 800 
4 None 27.3 E. coli 
160 5.9 
__________________________________________________________________________ 
.sup.a H. pylori and E. coli bacteria were adjusted to 8 .times. 10.sup.9 
cells/mL and added to twofold antibody dilutions in microtiter plates. 
Methylene blue (0.01%) was added to the bacterial diluent (0.01 M 
phosphate buffered saline pH 7.0) to enhance visualization of 
agglutinated bacteria. 
.sup.b Human rotavirus (HRV). 
The dominant antibody class found in colostrum is immunoglobulin type G 
(IgG) of the IGGI isotype subclass. These antibodies possess the physical 
properties described in Table 3. 
TABLE 3 
______________________________________ 
BIOPHYSICAL CHARACTERIZATION OF ANTIBODIES 
CONTAINED IN COLOSTRUM 
______________________________________ 
Dominant Immunoglobulin Class: 
IgGl 
Molecular Weight: 160,000 Daltons 
Sedimentation Coefficient (S.sub.20,w): 
6.7 s 
Extinction Coefficient (E .sub.280 1% ): 
12.2 
Isoelectric Point: 5.5-6.8 
Carbohydrate Content: 3% 
______________________________________ 
EXAMPLE 4 
Preparation of Feed Material 
Following assay, as described above, the BCW samples were added to 
commercially available Similac infant formula (Ross Laboratories) to yield 
a standard concentration of 17 mg IgGl/mL. Following combination the 
samples were heat treated and an antibiotic mixture added to result in the 
production of a material free of viable bacteria. This is a requirement 
for the gnotobiotic piglet model. The individual concentrations of the 
antibiotics were selected so as to be noninhibitory for Helicobacter 
pylori growth only. All other bacteria growth was inhibited. 
The control feed was Similac a containing the same type and amount of 
antibiotic mixture. 
EXAMPLE 5 
Experimental Animals 
A piglet litter of 13 animals was aseptically delivered and maintained in 
germ free incubators according to standard procedures for establishing 
germ free animals. 
From this litter 11 animals were randomly divided into two experimental 
groups: 
Group I: 5 Piglets fed control Similac (D nutrient only; and 
Group II: 6 Piglets fed Similac a containing Helicobacter pylori antibody. 
Both experimental groups were orally infected with Helicobacter pylori at 3 
days of age with 2.times.10.sup.9 CFU/ML following pretreatment with 
Cimetidine. 
Proof of Helicobacter pylori infection was established by means of ELISA 
assays to detect Helicobacter pylori-specific porcine antibodies in serum 
samples from each piglet as illustrated in Table 4. The data contained in 
this Table indicates that both groups of animals clearly develop 
significant antibody levels of all three immunoglobulin isotypes in their 
sera following infection as compared to preinfection sera. 
TABLE 4 
__________________________________________________________________________ 
ISOTYPE SPECIFIC ELISA TESTING OF INFECTED AND CONTROL 
GERMFREE PIGLET SERA FOR ANTIBODY TO H. pylori 
MEAN OPTICAL DENSITY 
PIGLET GROUP ISOTYPE SPECIFIC SERUM ANTIBODY 
AND TIME SAMPLED IgG IgM IgA 
__________________________________________________________________________ 
ALL PIGLETS &lt;0.01 &lt;0.02 &lt;0.03 
BEFORE INFECTION 
IMMUNE COLOSTRUM FED 
0.58 0.32 0.38 
AFTER INFECTION 
CONTROL (SIMILAC) FED 
0.77 0.38 0.35 
AFTER INFECTION 
__________________________________________________________________________ 
EXAMPLE 6 
Feeding Regimen 
Ten days following Helicobacter pylori infection, piglets were fed either 
Similac.RTM. only (Group I) or Similac.RTM. containing Helicobacter pylori 
specific antibodies (Group II) at a concentration of 17 mg IgGl/ml. The 
animals each received 30 mL of feed material three times each day. 
Following feeding, each animal received 150 mL of mild replacer diet which 
did not contain antibodies. The feeding protocol continued for a twenty 
day period. 
EXAMPLE 7 
Post Protocol Assessment 
At age 33 days, blood samples were drawn from each animal and the animals 
were euthanized and necropsied. The blood samples were processed to serum 
and analyzed for the presence of porcine anti-Helicobacter pylori 
antibodies. Gastric epithelium samples (1-2cm.sup.2) from five different 
anatomic regions were taken at necropsy and subsequently evaluated for 
bacterial colonization and histologic evidence of infection. The biopsies 
were taken from the cardiac, fundic, pyloric, antrum and diverticulum 
regions of the stomach. 
Biopsy samples were placed on selective agar plates containing the selected 
antibiotic mixture and streaked. The innoculated plates were incubated in 
gas jars in a reduced oxygen atmosphere consisting of 5% O.sub.2, 10% 
CO.sub.2 and 80% N.sub.2 at 37.degree. C. which is a gas mixture which 
selectively facilitates Helicobacter pylori growth. The plates were 
examined for bacterial growth after 5-8 days. Suspected Helicobacter 
pylori colonies were subcultured onto fresh medium. Gram stains were 
performed on both the bacterial growth as well as on the mucoid material 
associated with the biopsies. Identity of the bacteria was confirmed using 
standard enzymatic (catalase, oxidase, urease) and antibiotic sensitivity 
(Nalidixic acid and Cephalothin) assays. The profiles provided by these 
assays allow for the accurate definition of the type of bacteria being 
examined. This methodology is standard in the art. The results for these 
studies are summarized in Tables 5 and 6. 
Data on the reisolation of viable bacteria from the various piglet stomach 
regions examined is shown in Table 5. A comparison was made between 
piglets fed the immune product as compared to those receiving nonimmune 
nutrient only. 
In piglets fed non-immune nutrient, small bacterial colonies typical of 
Helicobacter pylori developed on the agar plates in 84% of the 5 stomach 
epithelium biopsy sites assayed. In comparison, piglets fed nutrient 
containing specific anti-Helicobacter pylori antibodies, colonies were 
observed in only 37% of the biopsies. Viable Helicobacter pylori bacteria 
were isolated from 100% of the control piglets (i.e. those animals 
receiving nonimmune nutrient) whereas viable Helicobacter pylori bacteria 
were isolated from only 50% of the piglets fed the immune product. 
Identity of the bacteria as being Helicobacter pylori was confirmed using 
biochemical assays. The differences are significant at the 95% confidence 
level using the one-sided Students "t" test or the nonparametric ranking 
approach (Wilcoxin test). 
TABLE 5 
__________________________________________________________________________