# Patent

## Title
Composition and method for controlling intestinal pathogenic organisms

## Abstract

An antigen composition for stimulating an immune response in an inoculated avian species to at least one intestinal pathogenic organism includes seven field strains of E. coli, Pseudomona aeruginosa, Aerobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Salmonella agona, and Salmonella Kentucky. The antigen composition can be used alone or in combination with a Marek's Disease vaccine to reduce shedding of E. coli and/or Salmonella bacteria.

## Background
This application claims the benefit of earlier filed U.S. patent application Ser. No. 11/737,483 filed on 19 Apr. 2007, now abandoned.


FIELD OF THE INVENTION
The invention pertains generally to composition for controlling intestinal pathogenic organisms in avian species and, more particularly, to a multivalent antigen for inducing immunity to specific bacterial diseases and/or to enhance immunity in an infected organism.
BACKGROUND OF THE INVENTION
Consumption of poultry products contaminated with Salmonella bacteria is a significant source of gastrointestinal infections in humans. For example, Salmonella enteritidis, especially phage type 4, has become more common in both poultry and humans since the early 1980's. The prevalence of Salmonella typhimurium, on the other hand, has remained relatively stable. However, the spread of the antibiotic-resistant strain DT104 in domestic flocks gives some reason for concern. Accordingly, the presence of Salmonella in commercial meat and food products is a major public health concern given that such infections can lead to serious illness or, in severe cases, death. Further, Salmonella infections in chickens, turkeys and ducks raise concerns for poultry producers due to increasing rates of morbidity and mortality as well as losses attributable culling and/or rejection of infected birds.
Salmonella infections can be spread via intraspecies or horizontal transmission, i.e., from animal to animal, and/or via interspecies or vertical transmission, i.e., from animal to humans. Generally, horizontal transmission of Salmonella bacteria is typically via exposure to environmental factors such as, for example, contaminated feces, bedding, nesting materials and/or other fomites. In contrast, vertical transmission of Salmonella bacteria is typically via oral exposure to the bacteria such by handling contaminated raw meats. Vertical transmission can also occur via shell contamination and/or internal transovarian contamination of the yolk of eggs produced by infected birds.
The basis for good control of Salmonella infections in farm environments, in particular, in poultry farms, is good farming and hygiene practices. Such practices include, for example, managing and preventing contamination of feeds, monitoring of animal health, cleaning and disinfection of coops and pens, and control of pest species such as, for examples, rodents. Testing and removal of infected or pathogen-positive animals from production and/or contact with uninfected animals are also vital to controlling horizontal and/or vertical transmission of such infections.
Poultry infected with Salmonella bacteria generally develop a strong immune response to the pathogen which is typically manifested by progressive reduction in excretion of the organism and reduced disease and excretion upon subsequent challenge. Accordingly, there is a need for an effective means for inducing an immune response to Salmonella bacteria in poultry which results in reduced disease and excretion or shedding of the bacteria while reducing productivity losses attributable to culling and/or rejection of infected birds.
Recently, vaccination of commercial poultry flocks to increase resistance against pathogenic exposure to Salmonella has become more prevalent particularly in view of increasing public awareness. However, such vaccination programs are generally difficult, time consuming and/or prohibitively expensive to administer on a commercial production scale. Accordingly, there is a need for an effective means for vaccinating domestic poultry and fowl against Salmonella infections.
Additionally, it is generally believed that vaccination is not a control option for serovars other than Salmonella enteritidis and Salmonella typhimurium which can be present on poultry farms. It is also generally believed that vaccination has limited effect on improving animal health and welfare and such vaccines are primarily used for public health reasons. Accordingly, there is a need for an antigen composition or vaccine effective to result in improved avian health and welfare such as can be manifested by increased weight gain and reduced mortality.
Further, some antigens may interfere with efficacy of other vaccines or medications administered simultaneously with and/or subsequent to vaccination. Additionally or alternatively, particular antigens may interfere with or affect the accuracy of traditional test or screening tools used to detect active or prior infection. Accordingly, there is a demand for a Salmonella antigen which can be administered to domestic poultry and fowl which does not reduce the effectiveness of other vaccines such as, for example, Marek's disease vaccines.
SUMMARY OF THE INVENTION
A general object of the invention is to provide a multivalent antigen for inducing an immune response and/or providing enhanced immunity to a pathogenic organism such as Salmonella spp.
A more specific object of the invention is to overcome one or more of the problems described above.
The general object of the invention can be obtained, at least in part, through a multivalent antigen composition comprising seven field strains of E. coli, Pseudomona aeruginosa, Aerobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Salmonella agona and Salmonella Kentucky. The composition induces an immune response in an inoculated avian species to at least one intestinal pathogenic organism.
The prior art generally fails to provide a Salmonella-containing multivalent antigen composition which is as effective as desired in inducing an immune response to at least one intestinal pathogenic organism such as, for example, Salmonella spp. which is manifested by a reduced fecal count in an inoculated avian species. The prior art further generally fails to provide a multivalent antigen composition which can be easily and effectively administered in a commercial farm environment at a reduced cost. The prior art additionally fails to provide a multivalent antigen composition that can be utilized alone or in combination with other vaccine products without reducing the efficacy of either vaccine component and/or the ability to detect or diagnose particular diseases within inoculated birds.
The invention further comprehends a bacterin vaccine comprising about 67% of seven field strains of E. coli, about 10% Pseudomona aeruginosa, about 10% Aerobacter aerogenes, about 4% Salmonella enteritidis, about 3% Salmonella typhimurium, about 3% Salmonella agona, and about 3% Salmonella Kentucky. The seven E. coli strains are selected from the group consisting of ATCC strain 25922, a University of Delaware field isolate, and five Delmarva field isolates.
The invention additionally comprehends an in ovo vaccine including a bacterin vaccine and a Marek's disease vaccine. The bacterin vaccine comprises seven field strains of E. coli, ATCC strain 27853 of Pseudomona aeruginosa, Aerobacter aerogenes, ATCC strain 13076 of Salmonella enteritidis, ATCC strain 14028 of Salmonella typhimurium, Salmonella agona, and Salmonella Kentucky. At least one field strain of E. coli consists of ATCC strain 25922 and each strain of E. coli is present in substantially equal amounts. The in ovo vaccine reduces a concentration of at least one pathogenic organism in a gastrointestinal tract of an inoculated avian species.
As used herein the term “bacterin” or “bacterin vaccine” refers to a vaccine composition generally comprised of dead or inactivated bacteria species.
As used herein the terms “about” and “substantially” when used in conjunction with a percentage or the term “equal” refer to a value falling within a range of ±1 percentage point. For example, a concentration of about 5% includes all concentrations falling within the range of 4% to 6%.
Other objects and advantages will be apparent to those skilled in the art from the following detailed description taken in conjunction with the examples and the appended claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a chart showing the effect of an antigen composition of the invention on Marek's Disease vaccine titration in culture.
FIG. 2 is a chart showing the effect of an antigen composition of the invention on Marek's Disease vaccine re-isolation titers in vivo.



DETAILED DESCRIPTION
The invention provides a multivalent antigen or antigen composition which stimulates an immune response in an inoculated avian species to at least one intestinal pathogenic organism. The multivalent antigen composition includes seven field strains of E. coli, Pseudomona aeruginosa, Aerobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Salmonella agona and Salmonella Kentucky. 
In accordance with certain embodiments, the multivalent antigen or antigen composition stimulates an immune response to an intestinal pathogenic organism selected from Clostridium perfringens, Salmonella spp., E. coli or a combination thereof. Such immune response can be manifested as a reduction in fecal bacterial counts for a particular pathogen such as, for example, reduction in Salmonella spp. fecal bacteria counts and/or E. coli fecal bacterial counts. Such immune response can additionally or alternatively be manifested as a reduction in lesion formation upon exposure to Clostridium perfringens. 
Various strains of E. coli bacteria can be included in the antigen composition. Suitably, such strains of E. coli bacteria can be selected from ATCC strain 25922, a University of Delaware field isolate, one or more Delmarva field isolates or a combination thereof. In accordance with one embodiment, the antigen composition includes seven field strains of E. coli bacteria including ATCC strain 25922, a University of Delaware field isolate and five Delmarva field isolates.
In accordance with certain embodiments, the antigen composition includes about 67% of seven strains of E. coli bacteria. Suitably, each strain of E. coli bacteria is present in an approximately equal amount.
Various strains of Pseudomona aeruginosa are suitable for use in the antigen composition. In accordance with one embodiment, the antigen composition can include ATCC strain 27653 of Pseudomona aeruginosa. Suitably, the antigen composition can include about 10% Pseudomona aeruginosa. 
The antigen composition further includes Aerobacter aerogenes such as in a concentration of about 10%. In accordance with certain aspects of the invention, the antigen composition is or should be free or devoid of Enterobacter aerogenes and/or Klebsiella pneumoniae. 
The antigen composition also includes at least four strains of Salmonella species. In particular, the antigen composition includes Salmonella enteritidis, Salmonella typhimurium, Salmonella agona and Salmonella Kentucky. In accordance with certain embodiments, the antigen composition can include ATCC strain 13076 of Salmonella enteritidis and/or ATCC strain 14028 of Salmonella typhimurium. 
Suitably, the antigen composition, in accordance with one embodiment, can include about 4% Salmonella enteritidis, about 3% Salmonella typhimurium, about 3% Salmonella agona and about 3% Salmonella Kentucky.
Suitably, the multivalent antigen or antigen composition can be utilized as or in an in ovo vaccine for inoculating avian species or domestic fowl. For example, about 0.005 ml to about 0.05 ml of the multivalent antigen or antigen composition can be used to inoculate an embryonated egg. In accordance with certain embodiments, the multivalent antigen or antigen composition can be given in a dose of about 0.0063 ml to about 0.0375 ml per embryonated egg.
The multivalent antigen or antigen composition is suitable for use alone as a bacterin vaccine or in combination with one or more other vaccine preparations. For example, the antigen composition can be administered sequentially with or simultaneously with another vaccine preparation such as, for example, a Marek's Disease vaccine.
In accordance with one embodiment, the antigen composition can be mixed or combined with a Marek's Disease vaccine. Such combined or mixed vaccine comprises a bacterin vaccine including seven strains of E. coli, Pseudomona aeruginosa, Aerobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Salmonella agona and Salmonella Kentucky and a Marek's Disease vaccine. The Marek's Disease vaccine can include an HVT vaccine, a SB-1 vaccine or a bivalent vaccine including a mixture or combination of HVT and SB-1 strains. Advantageously, the bacterin and Marek's Disease vaccine may be combined in any suitable ratio. For example, the combined vaccine may have a bacterin vaccine to Marek's Disease vaccine ratio in the range of about 1:15 to 15:1. In accordance with certain embodiments, the bacterin vaccine and the Marek's Disease vaccine can be combined in a 1:1 ratio.
The combined bacterin-Marek's Disease vaccine reduces a concentration of at least one pathogenic organism in a gastrointestinal tract of an inoculated avian species. Such pathogenic organism can include E. coli, Salmonella spp. or a combination thereof.
Suitably, the combined bacterin-Marek's Disease vaccine can be an in ovo vaccine suitable for inoculating an avian species or domestic fowl such as, for example, chickens, ducks, geese and/or turkeys. For example, the combined bacterin-Marek's Disease vaccine can be administered in ovo in a dose of about 0.005 ml to about 0.1 ml combined vaccine per embryonated egg. In accordance with certain embodiments, a dose of the combined bacterin-Marek's Disease vaccine can include about 0.0063 ml to about 0.0375 ml bacterin vaccine.
A method for reducing transmission of pathogenic gastrointestinal organisms includes inoculating an avian species in ovo at about 18 days embryonic age with the above-described antigen composition alone such as, for example, as a bacterin vaccine or in combination with another vaccine preparation such as, for example, a Marek's Disease vaccine.
EXAMPLES
Antigen Composition:
An antigen composition was produced using various strains of bacteria, shown in TABLE 1, below, commonly found in poultry and/or humans. Each bacteria isolate was initially individually grown in 1000 ml of Nutrient Broth (Sigma N7519) at 35±1° C. for 24±2 hours. After the incubation period, each broth was centrifuged for approximately 10 minutes at 5000 rpm in individual centrifuge sectors to separate the cells from the broth. The supernatant was then aseptically removed from each centrifuge vessel. The remaining cultures from each tube were then re-suspended in Butterfield's Phosphate diluent and tested to determine purity. The purified cultures collectively formed a Master Seed.
The above steps were repeated until the quantity of Master Seed required to produce mass quantities of Working Seed stock was achieved. Following determination of purity and specie, all Working Seed stock batches were mixed, separated into batch fermentation vessels and grown at 35±1° C. for 24±2 hour periods. At the completion of each batch, the entire batch was carefully mixed and a sample of each culture was then plated onto Nutrient Agar. Colonies were counted after a further incubation period of 24±2 hours at 35±1° C. using 10-fold dilutions up to 1010 dilution rate. Plates with CFUs between 30 and 300 were counted.










TABLE 1







Bacterial component
CFU Counts









E. coli Isolate #1
1.36 × 1010



E. coli Isolate #2
2.03 × 1010



E. coli Isolate #3
6.80 × 109



E. coli Isolate #4
2.92 × 1010



E. coli Isolate #5
1.28 × 1010



E. coli Isolate #6
2.13 × 1010



E. coli Isolate #7
5.30 × 1010



Pseudomona aeruginosa
2.14 × 109



Aerobacter aerogenes
9.40 × 108



Salmonella enteritidis
1.86 × 109



Salmonella typhimurium
2.38 × 109



Salmonella agona
4.10 × 109



Salmonella Kentucky
6.50 × 109










The final counts were used to dilute and mix the individual cultures into the final antigen composition or bacterin vaccine, as shown in TABLE 2, below. The bacteria were then killed by autoclaving at 121° C. for 15±2 minutes. This procedure was then repeated to ensure total bacteria kill.










TABLE 2







Bacterial Component
Concentration









E. coli (7 field strains)
67% (each strain in ≈ equal amounts)



Pseudomona Aeruginosa
10%



Aerobacter aerogenes
10%



Salmonella enteritidis
4%



Salmonella typhimurium
3%



Salmonella agona
3%



Salmonella Kentucky
3%










Effect on Lesion Formation Due to Clostridium perfringens Exposure
Fourteen treatment groups of Ross (Male)×Cobb (Female) broilers were in ovo inoculated with either a saline control or the antigen composition described in TABLE 2 at an embryonic age of 18 days. The fourteen treatment groups include seven (7) control groups each including 20 male and 20 female chicks and seven (7) vaccine groups each including 20 male and 20 female chicks.
All birds were inoculated with Clostridium perfringens (104 per bird) on post-hatch Day 8 to induce necrotic enteritis. Four male and four female birds from each control group and each vaccine group were humanely euthanized on Days 21 and 49, necropsied and the intestinal tracts visually inspected for signs of necrotic enteritis and/or coccidiosis. The intestinal lesion scores including both coccidiosis signs and necrotic enteritis signs were recorded. Lesions were scored on a scale of 0 to 4 as follows:

    
    
        0=No lesions found;
        1=Slight redness with no cell sloughing (mucus);
        2=Moderate redness and/or slight cell sloughing;
        3=Severe redness and/or severe cell sloughing; and
        4=Actual bleeding observed.
    
    


The lesion score data, as summarized in TABLE 3, below, indicate that birds inoculated with the above-described antigen composition or bacterin vaccine had a statistically lower rate of development of intestinal lesions between Days 8 and 21. Lesion scores recorded on Day 49 for both the control and vaccinated populations were not significantly different. Accordingly, it is believed that inoculation with the antigen Composition of the invention induces an immune response to the intestinal pathogen Clostridium perfringens thereby reducing the incidence, duration and/or severity of necrotic enteritis in avian populations.
Effect on Preharvest Intestinal Crop Bacteria.
All of the birds from the fourteen treatment groups described above were additionally inoculated with Escherichia coli (106 per bird) and Salmonella spp. (104 per bird) administered via oral gavage on post-hatch Day 15. Eight birds (4 males and 4 females) from each control and each vaccine group were humanely euthanized and necropsied on post-hatch Days 21 and 49. Crop content bacteria from each bird were plated on an appropriate agar and fecal E. coli and Salmonella spp. bacteria counts were recorded.
The fecal bacteria data, summarized in TABLE 3, below, indicate that birds inoculated with above-described antigen composition or bacterin vaccine had a statistically lower concentration of both E. coli and Salmonella bacteria present in the crop contents. Accordingly, it is believed that inoculation with the antigen composition of the invention induces or stimulates an immune response to E. coli and Salmonella resulting in reduced fecal bacteria content as well as reduced shedding of bacteria.
Additionally, it was determined, as summarized in TABLE 3, below, that birds inoculated with the above-described antigen composition exhibited an increase in average weight gain over the duration of the 49 day study.










TABLE 3









Day 21
Day 49











Criterion
Control
Vaccine
Control
Vaccine














Average Lesion Score
1.018
0.250
0.179
0.107


Std Dev.
0.29
0.09
0.15
0.10


C.V.
28.93
37.80
82.46
97.18


Fecal E. coli count (per ml)
2085.0
786.2
1479.9
532.1


Std Dev.
158.80
138.90
161.02
67.59


C.V.
7.62
17.67
10.88
12.70


Fecal Salmonella spp.
160.0
122.4
129.7
94.7


count (per ml)


Std Dev.
16.85
11.10
12.86
9.98


C.V.
10.53
9.07
9.92
10.54


Average Weight Gain (g)
547.648
580.171
2146.721
2225.416


Std Dev.
7.13
7.97
58.50
59.18


C.V.
1.30
1.37
2.72
2.66









Effect on Marek's Disease Vaccine
A study was conducted to determine if the above-described antigen composition or bacterin vaccine if administered in combination with commercially available Marek's Disease vaccine negatively impacted the replication of the vaccine viruses in cell culture or in vivo. Such a negative impact, as determined by decreases in the ability to re-isolate vaccine viruses at one week post-hatch, would suggest that the antigen composition may decrease Marek's Disease vaccine efficacy.
Effect on Marek's Disease Vaccine in Culture.
To assess the effect of the above-described antigen composition on Marek's Disease vaccine preparations, the antigen composition and its diluent were obtained at 4× concentration. These were added to 4× stocks of HVT and SB-1 to generate 2× stocks of HVT and SB-1. Upon mixing of equal amounts, this yielded 1× bivalent vaccines containing either 1× diluent or 1× antigen composition.
The vaccine stocks were titrated independently from the 4× stocks and also titrated from each of the 1× final stocks. This was to determine the effect of the antigen composition on HVT and SB-1 replication, in culture and to determine if the antigen composition would interfere with titration of commercial vaccine. In each case, a commercial diluent was used for diluting the vaccines. Vaccine, viruses and diluent were obtained from commercial sources.
As indicated by the titration data, summarized in TABLE 4, below, and shown in FIG. 1, the antigen composition did not negatively affect Marek's Disease replication in cell culture. Titration of the vaccine stocks after either diluent or antigen composition addition showed essentially identical titers.













TABLE 4










Bird Dose
Std


Vaccine
PFU/Vial
Dose
Dilution
Mean Plaque #
(PFU)
Dev.





















HVT
1.59 × 107
4X
1:50
120.8 (±8.5)
6040
1028


SB-1
 4.2 × 105
4X
1:50
  123 (±14)
3075
742


HVT +

1X
1:100
51.75 (±7.5)
5175
750


diluent


SB-1 +

1X
1:100
20.75 (±6.4)
2075
640


diluent


HVT +

1X
1:100
 53.3 (±3.9)
5325
386


antigen


SB-1 +

1X
1:100
 26.5 (±3.9)
2650
387


antigen









Effect on Marek's Disease Vaccine In Vivo
Eggs from a commercial broiler chicken strain, Ross X Cobb breed, were inoculated at 18 days embryonic age with either a bivalent HVT/SB-1 Marek's Disease vaccine (5000 PFU/bird HVT+2500 PFU/bird SB-1) mixed with a control diluent (vaccine+diluent) or a vaccine including the bivalent Marek's Disease vaccine mixed with the above-described antigen composition (vaccine+antigen). Post-hatch, an equal number of male and female chicks were randomly placed in grow out pens and grown under practical commercial conditions.
At one week post-hatch chickens were bled via cardiac puncture, euthanized and the spleens were pooled into groups. The vaccine+diluent and vaccine+antigen groups were each comprised of four (4) pools of three (3) birds.
Blood and spleens were pooled and PBMC were purified from the whole blood by histopaque centrifugation. Spleen cells were washed, counted and plated at 2×10 cells in triplicate dishes for each pool. PBMC were not co-cultivated with CEF monolayers, as HVT and SB-1 infection is characteristically low at this time. At six (6) days post-plating, the dishes were examined and plaques for HVT and SB-1 were counted.
The above procedure was repeated three times over the course of four (4) weeks, i.e., a total of 16 pools of birds from the vaccine+diluent and a total of 16 pools of birds from the vaccine+antigen groups were inoculated and evaluated. The data obtained from the re-isolation counts were subjected to Chi-square and Students t-test analysis, the results of which are summarized in TABLE 5, below, and shown in FIG. 2.








TABLE 5







Vaccine + Diluent
Vaccine + Antigen












Group #
Strain
Count
Group #
Strain
Count





1A
HVT
48 ± 1
1B
HVT
47 ± 13



SB-1
21 ± 3

SB-1
13 ± 1


2A
HVT
58 ± 14
2B
HVT
56 ± 22



SB-1
16 ± 3

SB-1
19 ± 2


3A
HVT
67 ± 16
3B
HVT
37 ± 2



SB-1
17 ± 4

SB-1
15 ± 2


4A
HVT
42 ± 1
4B
HVT
69 ± 8



SB-1
25 ± 5

SB-1
20 ± 1










HVT Overall Average
54
HVT Overall Average
52


SB-1 Overall Average
20
HVT Overall Average
17









The results in TABLE 5 indicate that comparable counts of HVT and SB-1 plagues were obtained from the two treatment groups and, thus, overall no significant differences were found for either the HVT or the SB-1 data.
In Week 4 of the study, a statistically significant difference was found in the HVT counts between the vaccine+diluent and the vaccine+antigen groups. The antigen was found to increase the titers of HVT re-isolated from inoculated chickens at one-week post-hatch. This is believed to indicate an advantage conferred on the replication of HVT. Conversely, a small but statistically significant difference was found between SB-1 re-isolated from the inoculated chickens.
Overall, the bacterin vaccine or antigen composition did not negatively affect Marek's Disease replication in vivo. Thus, it is unlikely that the antigen composition would decrease the efficacy of Marek's Disease vaccines if employed in an in ovo vaccination program. Moreover, the addition of the antigen composition should not negatively affect the ability to titer vaccines.
While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.
The bacterial components, including E. coli Isolates #1-#7, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Salmonella agona, and Salmonella Kentucky, utilized in the formulation of the multivalent antigen compositions, in ovo vaccines, and bacterin vaccines disclosed herein were deposited with the American Type Culture Collection (ATCC®), Patent Depository, 10801 University Boulevard, Manassas, Va. 20110, on Jun. 8, 2010, were accepted on Jun. 22, 2010 and have been assigned Patent Deposit Designation (ATCC Deposit No.) PTA-11029. The Aerobacter aerogenes bacterial component utilized in the formulation of the multivalent antigen compositions, in ovo vaccines, and bacterin vaccines disclosed herein were deposited with the American Type Culture Collection (ATCC®), Patent Depository, 10801University Boulevard, Manassas, Va. 20110, on Feb. 3, 2011, were accepted on Feb. 25, 2011 and have been assigned Patent Deposit Designation (ATCC Deposit No.) PTA-11661.

## Claims

1. A multivalent antigen composition, comprising a suspension of dead or inactivated bacteria including:
(a) seven field strains of E. coli, each field strain present in an approximately equal amount;
Pseudomonas aeruginosa; 
Salmonella enteritidis; 
Salmonella typhimurium; 
Salmonella agona; and
Salmonella Kentucky, 

said bacteria having ATCC Deposit No. PTA-11029; and
(b) Aerobacter aerogenes; 
the multivalent antigen composition stimulating an immune response in an avian species inoculated with said multivalent antigen composition to at least one intestinal pathogenic organism selected from the group consisting of Clostridium perfringens, Salmonella species, Escherichia coli, and combinations thereof.

2. The multivalent antigen composition of claim 1, wherein the multivalent antigen composition is in ovo antigen vaccine.

3. The multivalent antigen composition of claim 1, further comprising a Marek's disease vaccine.

4. The multivalent antigen composition of claim 3, wherein the suspension of dead or inactivated bacteria and the Marek's disease vaccine are present in a ratio of 1:1.

5. The multivalent antigen composition of claim 1, wherein the suspension of dead or inactivated bacteria is prepared by:
separately culturing each bacteria at temperature of 35±1° C. for a period of 24±2 hours;
sampling and plating each culture onto individual Nutrient Agar plates;
incubating the plated cultures for a period of 24±2 hours at a temperature of about 35±1° C.;
counting bacterial colonies on the individual Nutrient Agar plates;
mixing the individual bacteria cultures into an antigen composition in a select ratio based on the colony counts; and
autoclaving the antigen composition at 121° C. for 15±2 minutes.

6. A bacterin vaccine, comprising:
(a) about 67% of seven field strains of E. coli, each strain of E. coli present in an approximately equal amount;
about 10% Pseudomonas aeruginosa; 
about 4% Salmonella enteritidis; 
about 3% Salmonella typhimurium; 
about 3% Salmonella agona; and
about 3% Salmonella Kentucky,

said bacteria having ATCC Deposit No. 11029; and
(b) about 10% Aerobacter aerogenes; 
wherein the bacterin vaccine is prepared by:
separately culturing each bacteria at temperature of 35±1° C. for a period of 24±2 hours;
sampling and plating each culture onto individual Nutrient Agar plates;
incubating the plated cultures for a period of 24±2 hours at a temperature of about 35±1° C.;
counting bacterial colonies on the individual Nutrient Agar plates;
proportionately mixing the individual bacteria cultures into an antigen composition based on the colony counts; and
autoclaving the antigen composition at 121° C. for 15±2 minutes.

7. A method for reducing transmission of pathogenic gastrointestinal organisms, comprising:
inoculating an avian species in ovo at about 18 days embryonic age with the vaccine according to claim 6.

8. An in ovo vaccine, comprising:
a bacterin vaccine including:
(a) seven field strains of E. coli, wherein each strain of E. coli is present in substantially equal amounts,
Pseudomonas aeruginosa, 
Salmonella enteritidis, 
Salmonella typhimurium, 
Salmonella agona, and
Salmonella Kentucky; 

said bacteria having ATCC Deposit No. 11029, and
(b) Aerobacter aerogenes; and

a Marek's disease vaccine,
the in ovo vaccine reducing a concentration of at least one pathogenic organism selected from the group consisting of E. coli spp., Salmonella spp., or a combination thereof in a gastrointestinal tract of an avian species inoculated in ovo with said vaccine.

9. The in ovo vaccine of claim 8, wherein the Marek's disease vaccine is selected from the group consisting of HVT vaccines, SB-1 vaccines, and combinations thereof.

10. The in ovo vaccine of claim 8, wherein the avian species is a domestic fowl selected from the group consisting chickens, ducks, geese and turkeys.

11. A method for reducing transmission of pathogenic gastrointestinal organisms, comprising:
inoculating an avian species in ovo at about 18 days embryonic age with the vaccine according to claim 8.

12. The in ovo vaccine of claim 8, wherein the bacterin vaccine is prepared by:
separately culturing each bacteria at temperature of 35±1° C. for a period of 24±2 hours;
sampling and plating each culture onto individual Nutrient Agar plates;
incubating the plated cultures for a period of 24±2 hours at a temperature of about 35±1° C.;
counting bacterial colonies on the individual Nutrient Agar plates;
mixing the individual bacteria cultures into an antigen composition in a select ratio of about 67:10:10:4:3:3:3: based on the colony counts; and
autoclaving the antigen composition at 121° C. for 15±2 minutes.

13. The in ovo vaccine of claim 8, wherein the bacterin vaccine and the Marek's disease vaccine are combined in a ratio of from about 1:15 to about 15:1.

14. The in ovo vaccine of claim 13, wherein a dose comprises from about 0.005 ml to about 0.1 ml if the in ovo vaccine.