U.S. patent application number 11/658397 was filed with the patent office on 2009-12-03 for method for vaccination of poultry by bacteriophage lysate bacterin.
This patent application is currently assigned to Intralytix. Invention is credited to Torrey Brown, Gary R. Pasternack, Alexander Sulakvelidze.
Application Number | 20090297561 11/658397 |
Document ID | / |
Family ID | 34272802 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090297561 |
Kind Code |
A1 |
Pasternack; Gary R. ; et
al. |
December 3, 2009 |
Method for vaccination of poultry by bacteriophage lysate
bacterin
Abstract
The invention provides methods of generating phage lysate
bacterins, as well as phage lysate bacterin compositions. The
invention further encompasses methods of vaccination comprising
administering phage lysate bacterin to an animal in need thereof.
The invention further encompasses methods of reducing infection or
colonization of poultry or poultry eggs using phage bacterin
lysates. Method of vaccination comprising administering to an
animal in need of immunization an amount of phage lysate bacterin
to induce an immune response.
Inventors: |
Pasternack; Gary R.;
(Baltimore, MD) ; Sulakvelidze; Alexander;
(Towson, MD) ; Brown; Torrey; (Severna Park,
MD) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W., SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
Intralytix
Baltimore
MD
|
Family ID: |
34272802 |
Appl. No.: |
11/658397 |
Filed: |
September 3, 2004 |
PCT Filed: |
September 3, 2004 |
PCT NO: |
PCT/US2004/028776 |
371 Date: |
June 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60499339 |
Sep 3, 2003 |
|
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|
Current U.S.
Class: |
424/257.1 ;
424/234.1; 424/258.1; 435/235.1 |
Current CPC
Class: |
A61K 2039/70 20130101;
Y02A 50/30 20180101; Y02A 50/482 20180101; C12N 7/00 20130101; A61P
43/00 20180101; C12N 2710/16334 20130101; A61K 2039/552 20130101;
A61K 39/12 20130101; A61K 39/0275 20130101 |
Class at
Publication: |
424/257.1 ;
424/234.1; 424/258.1; 435/235.1 |
International
Class: |
A61K 39/108 20060101
A61K039/108; A61K 39/02 20060101 A61K039/02; A61K 39/112 20060101
A61K039/112; C12N 7/00 20060101 C12N007/00 |
Claims
1. A method of vaccination comprising administering to an animal in
need of immunization an amount of phage lysate bacterin sufficient
to induce an immune response in said animal.
2. The method of claim 1, wherein the bacterin is administered
orally.
3. The method of claim 17 wherein the phage lysate bacterin is
administered in a plurality of sequential doses.
4. The method of claim 3, wherein the plurality of sequential doses
is administered over a period of one to ten weeks.
5. The method of claim 1, wherein the phage lysate bacterin is
administered in ovo.
6. An isolated phage lysate bacterin derived from a bacterial
species, comprising bacteriophage particles and bacterial fragments
produced by phage lysis, the composition substantially free of
whole, live bacteria.
7. The isolated phage lysate bacterin of claim 6, derived from one
or more pathogenic E. coli strains.
8. The isolated phage lysate bacterin of claim 7, wherein the E.
coli strains comprise the K88 and F18 serotypes.
9. The isolated phage lysate bacterin of claim 6, derived from one
or more pathogenic Yersinia species.
10. The isolated phage lysate bacterin of claim 9, wherein the
Yersinia species comprise Yersinia pseudotuberculosis and Yersinia
enterocolitica.
11. (canceled)
12. (canceled)
13. A method for making phage lysate bacterin comprising
inoculating a suspension of live bacteria with a bacteriophage
lytic for the bacteria and incubating the inoculated suspension
until a substantial portion of the bacteria in the suspension are
lysed.
14. The method of claim 13, further comprising removing
substantially all whole bacteria from the suspension to produce the
bacterin.
15. The method of 13, wherein said bacterin is derived from one or
more pathogenic E. Coli strains.
16. The method of 13, wherein said bacterin is derived from one or
more pathogenic Yersinia species.
17. The method of 13, wherein said bacterin is derived from one or
more pathogenic Salmonella strains.
18. The method of 1, wherein said bacterin is derived from one or
more pathogenic E. coli strains.
19. The method of 1, wherein said bacterin is derived from one or
more pathogenic Yersinia species.
20. The method of 1, wherein said bacterin is derived from one or
more pathogenic Salmonella strains.
21. The bacterin of claim 6, wherein said bacterin is derived from
one or more pathogenic Salmonella strains.
22. The bacterin of claim 21, wherein said pathogenic Salmonella
strain is Salmonella enteriditis, Salmonella agona, Salmonella
heidelberg, Salmonella kentuckii, Salmonella hadar, Salmonella
newport, Salmonella thomson, and Salmonella typhimurium.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention is directed to the field of
vaccination for treatment or prevention of bacterial disease. In
particular, it is directed to a method of vaccinating poultry using
bacteriophage-induced bacterial lysates.
[0003] 2. Description of Related Art
Salmonella in Chickens
[0004] USDA estimates that in 50-75% of human salmonella cases the
microorganism is acquired from meat, poultry, or eggs, with poultry
serving as the primary vehicle of transmission. Salmonella are part
of the normal, colonizing intestinal flora in many animals,
including chickens. Studies conducted in the early 1990's by USDA
indicated that 20-25% of broiler carcasses and 18% of turkey
carcasses were contaminated with Salmonella prior to sale. See Food
Safety and Inspection Service (1995); 9 CFR Part 308; Pathogen
Reduction; Hazard Analysis and Critical Control Point (HACCP)
Systems; Proposed Rule; 60 Fed. Reg. 6774-6889.
[0005] According to the CDC FoodNet/Salmonella surveillance system,
the five most common human Salmonella isolates in the United States
during 1990-1995 were S. typhimurium, S. enteritidis, S.
heidelberg, S. newport, and S. Radar. Further, according to the
USDA/FSIS data, the five most common Salmonella serotypes isolated
from broiler chickens during the same period were S. heidelberg, S.
kentuckii, S. hadar, S. typhimurium, and S. thomson.
[0006] There are strong public health, regulatory, and trade
incentives for producers to reduce levels of Salmonella
contamination in poultry. In addition to human disease, microbial
contamination of animals that are very susceptible to microbial
pathogens often leads to disease and increased animal morbidity. In
commercial animal growing operations where animals may be crowded
in facilities where other animals have been previously raised, the
likelihood of such contamination is often great. This is
particularly true of salmonella in the poultry industry. See
Suzuki, S., "Pathogenicity of Salmonella enteriditis in poultry.
International Journal of Food Microbiology 21:89-105 (1994).
Infection with Salmonella species may cause significant disease in
poultry flocks. Suzuki notes that non-typhoid types of Salmonella
including S. enteritidis and S. typhimurium cause both overt and
symptomless infections in poultry. Acute outbreaks may occur in
young birds and under stress conditions with mortality rates
approaching 20% in some cases and stunting of chicks of affected
flocks. S. enteritidis may cause pericarditis, necrotic hepatic
foci, induration of the yolk sack remnant, various ovarian
abnormalities, peritonitis, and possible renal involvement.
Consistent with this, organisms can be recovered from heart, liver,
spleen, caeca, yolk sac, ovary, oviduct, peritoneum, egg, and feces
of infected birds. As another example, Muir et al. ("Comparison of
Salmonella typhimurium challenge models in chickens," Avian
Diseases, 42:257-264, 1998) compared two different models of avian
Salmonellosis using oral challenge or spread from infected litter.
In both cases, S. typhimurium was readily recovered from both
spleen and liver.
[0007] Protection from colonization by Salmonella requires
induction of an adaptive immune response involving the mucosal
immune system. The first three weeks of life are a well-known
critical period during which newly hatched chicks are at risk for
colonization by Salmonella (Smith, "The development of the flora of
the alimentary tract in young animals," Journal of Pathology and
Bacteriology, 89:95-122, 1965; Barnes, et al., "The intestinal
flora of the chicken in the period 2 to 6 weeks of age with
particular reference to the anaerobic bacteria," British Journal of
Poultry Science, 13:311-326, 1972). The specific, adaptive mucosal
immune response mediated by gut-associated lymphoid tissue is
thought to be a critical determinant of whether or not colonization
occurs (Fukotome, et al., "Intestinal mucosal immune response in
chickens following intraocular immunization with
liposome-associated Salmonella eizterica serovar enteritidis
antigen," Developmental & Comparative Immunology, 25:475-484,
2001; Muir, et al., "Immunity, vaccination and the avian intestinal
tract," Developmental & Comparative Immunology, 24:325-342,
2000).
[0008] Vaccination via the avian intestinal tract produces an
adaptive mucosal immune response. A number of recent studies
demonstrate that oral immunization of chickens produces an adaptive
immune response to the pathogen targeted by the vaccine. Allen et
al., for example, using an attenuated S. typhimurium strain,
demonstrated that oral immunization resulted in an IgA response by
B cells in the intestinal lamina propria and in the spleen (Allen,
et al., "Kinetics of the mucosa antibody secreting cell response
and evidence of specific lymphocyte migration to the lung after
oral immunization with attenuated S. enterica var. typhimurum,"
FEMS Immunology and Medical Microbiology, 27:275-281, 2000).
Fukotome, et al., demonstrated that intraocular immunization with a
liposomal Salmonella antigen preparation produced a secretory
immune response in the intestine that was capable of inhibiting the
adherence of S. enteritidis to HeLa cells in a model of the
attachment that occurs in intestinal colonization. Immunization
with Salmonella can protect against colonization, as shown in the
studies of Van Immerseel, et al. ("The effect of vaccination with a
Salmonella enteritidis aroA mutant on early cellular responses in
caecal lamina propria of newly-hatched chickens," Vaccine,
20:3034-3041 2002). They showed that administration of a live
mutant vaccine resulted in protection along with colonization of
liver and spleen. In reviewing strategies for induction of mucosal
immunity, Muir et al., described studies that used attenuated live
Salmonella typhimurium as a vaccine to protect chickens against
pathogenic Salmonella species (see Curtiss, et al., "Nonrecombinant
and recombinant avirulent Salmonella vaccines for poultry,"
Veterinary Immunology and Immunopathology, 54:365-372, 1996;
Hassan, et al, "Development and evaluation of an experimental
vaccination program using a live avirulent Salmonella typhimurium
strain to protect immunized chickens against challenge with
homologous and heterologous Salmonella serotypes," Infection and
Immunity, 62:5519-5527, 1994).
[0009] Immunization in ovo has proven very successful for agents
such as Newcastle Disease virus (see Muir, et al.), but has seen
little use for bacterial agents. Perhaps this is attributable to
the variable immune response known to occur following oral
immunization, which may, in fact, sometimes result in a suppressive
response rather than a beneficial one. One instance where in ovo
vaccination has proven to be effective is in the study of Noor and
co-workers ("In ovo oral vaccination with Campylobacter jejuni
establishes early development of intestinal immunity in chickens,"
British Journal of Poultry Science, 36:563-573, 1995), who injected
heat-killed Campylobacter into the amniotic fluid on day 16 of
incubation, followed by an oral booster in some animals on day 7
post hatch. This procedure resulted in a striking specific
intestinal IgA response.
[0010] To date, vaccination approaches to reduction of Salmonella
colonization have achieved only partial success. Approaches to
Salmonella vaccination have employed attenuated strains (Alderton,
et al., "Humoral responses and Salmonellosis protection in chickens
given a vitamin-dependent Salmonella typhimurium mutant, Avian
Diseases, 35:435-442, 1991; Hassan. et al., (1993) "Effect of
infective dose on humoral immune responses and colonization in
chickens experimentally infected with Salmonella typhimurium,"
Avian Diseases, 37:19-26), or preparations of Salmonella antigens
prepared by sonication and detergent extraction of bacteria
(Hassan, et al., (1993); Methner, et al., "Comparative study of the
protective effect against Salmonella colonization in newly hatched
chicks using live attenuated Salmonella vaccine strains, wild-type
Salmonella strains, or a competitive exclusion product,"
International Journal of Food Microbiology, 35:223-230, 1993).
Other immunologic approaches have employed vaccination of breeder
flocks to promote maternal antibody transfer (Methner, et al.,
"Wirksamrkeit maternaler Salmonellerantikorper gegen eine orale
Testinfektion von Kuken mit Salmonella enteritidis," Berliner und
Munchener tierarztliche Wochenschrift, 110:373-377, 1997). It is
likely that the nature of the antigen, the route of administration
and anatomic localization of the adaptive immune response, and the
timing of the immunization are all critical factors. Of note, there
is one report in the patent literature where inoculation in ovo
with a bacteriophage increased the hatch rate of eggs
simultaneously infected with Salmonella typhimurium (Taylor, et
al., U.S. Pat. No. 2,851,006). This study did not, however, examine
the chicks for levels of colonization.
[0011] The scientific literature describes the use of an oral
Salmonella bacterin administered in ovo and post-hatch to induce an
adaptive immune response and reduce the percentage of birds
colonized by Salmonella species by acting primarily through the
mucosal immune system. However, there remains a need for improved
methods of protecting animals, especially poultry, against
bacterial colonization and/or infection by vaccination.
Vaccination in ovo
[0012] The desirability of injecting materials into avian eggs
during incubation has been recognized for some time. Initially, the
purpose of injecting eggs was to prepare various vaccines using the
egg as a growth medium for the vaccine. More recent developments
have involved injecting live embryonated eggs for the purpose of
accomplishing some beneficial or therapeutic effect on the embryo
or the bird that eventually hatches from the egg. Such beneficial
effects include increased growth, decrease post-hatch mortality
rates, increase the potential growth rates or eventual size of the
resulting chicken, disease resistance due to in ovo vaccination,
increased percentage hatch of incubated eggs, and otherwise
improved physical characteristics of hatched poultry.
[0013] Examples of substances which have been proposed as viable
treatment (or harvestable vaccine material) alternatives for
delivery via in ovo injection of avian embryos include live culture
vaccines, antibiotics, vitamins, and even competitive exclusion
media (a live replicating organism). Specific examples of treatment
substances are described in U.S. Pat. No. 4,458,630 to Sharma et
al, and U.S. Pat. No. 5,028,421 to Fredericksen et al.
[0014] Several basic techniques for injecting materials into live
embryonated eggs have been described, including forcing fluids
through the egg shell using pressurization and physically forming
an opening in the shell of an egg and then adding the desired
material (e.g., injection using syringe and needle arrangements).
One traditional method has been syringe injection of eggs by
hand.
[0015] In order to deliver material into eggs in a routine manner,
particularly in commercial egg production, it would be preferable
to employ some type of automated injection devices. For example, a
number of automatic devices have been disclosed for injecting eggs.
These include patents to Sandhage, U.S. Pat. No. 3,377,989 and to
Miller, U.S. Pat. Nos. 4,040,388; 4,469,047; and 4,593,645, and to
Hebrank U.S. Pat. No. 4,681,063. Sandhage discloses a hand operated
egg injection device for injecting a few eggs at the same time, but
does not disclose any method or system for handling large numbers
of eggs quickly and accurately. Miller '388 discloses an automated
apparatus for injecting the smaller ends of eggs and resealing the
holes produced, and Miller '047 shows a somewhat different
automated device for injecting eggs from their larger, air sac
ends. Hebrank '063 discloses an automated injection system for
embryos within eggs which has an advanced fluid delivery system
which eliminates the pumping of fluids through conventional fluid
handling systems and thereby reduces or eliminates the possibility
of the contamination of the fluid and provides a more accurate
volume delivery of such fluids. There exist other alternative
devices for automated injection of eggs as described more fully
below.
[0016] There continues to be a pressing need to eliminate
infections in poultry production both for the health benefits to
the bird itself as well as to decrease the likelihood of
transmitting pathogenic microorganisms through the food chain
eventually to the consumer of retail chicken and turkey products.
However, prior to the present invention the problems associated
with all other methods of decreasing flock contamination have not
been solved. Examples of such problems include the use of
antibiotics in feeds and the cost of environmental decontamination
of poultry production facilities.
SUMMARY OF THE INVENTION
[0017] It is an object of this invention to provide an improved
immunogenic composition for use in vaccination of animals,
including mammals and birds.
[0018] This and other objectives are addressed by one or more of
the following embodiments.
[0019] In one embodiment, this invention provides a method of
vaccination comprising administering to an animal in need of
immunization an amount of phage lysate bacterin sufficient to
induce an immune response in said animal. In a preferred mode, the
bacterin is administered orally. The phage lysate bacterin may be
administered in a plurality of doses sequentially, and the
sequential doses may be spaced over a period of one to ten weeks.
In another preferred mode, at least one dose of said bacterin is
administered in ovo.
[0020] In another embodiment, this invention provides a phage
lysate bacterin comprising bacteriophage particles and bacterial
fragments produced by phage lysis, said bacterin being
substantially free of whole, live bacteria.
[0021] In still another embodiment, this invention provides a phage
lysate bacterin produced by inoculating a suspension of live
bacteria with a bacteriophage lytic for the bacteria and incubating
the inoculated suspension until substantially all of the bacteria
in the suspension are lysed. Alternatively, the phage lysate
bacterin is produced by inoculating a suspension of live bacteria
with a bacteriophage lytic for the bacteria and incubating the
inoculated suspension until a substantial portion of the bacteria
in the suspension are lysed, then removing substantially all whole,
live bacteria from the suspension to produce the bacterin.
[0022] In yet another embodiment, this invention provides a method
of treating hatchery eggs to reduce Salmonella infection in a flock
comprising the steps of (1) providing a phage lysate bacterin
comprising at least one bacteriophage and (2) injecting said
bacterin into a fertilized egg under conditions appropriate to
decrease or eliminate colonization of the bird hatched from the egg
by Salmonella microorganisms. Introduction of the bacterin may be
carried out by injecting the bacterin in ovo into any compartment
of the egg, including the body of the embryo. Typically, the egg
into which the bacterin is introduced is incubated to hatch.
[0023] In still another embodiment, this invention provides a
method of treating poultry to reduce bacterial colonization of a
flock comprising the steps of (1) providing a phage lysate bacterin
comprising at least one bacteriophage and (2) treating birds with
the bacterin under conditions which enable the bacterin to be
effective in causing a decrease or elimination of colonization of
the birds by pathogenic microorganisms. The birds may be treated
with the phage lysate bacterin by oral dosing in drinking water, by
injection, or by spraying. The birds may be treated with bacterin
at any age, such as within the first five days following hatching,
on the day of hatch, or the bacterin may be delivered to the eggs
by injection, including by automated injection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a graph comparing the reduction in Salmonella
contamination obtained at three weeks of age by injection of eggs
with bacteriophage lysate followed by spraying of chicks with the
lysate.
[0025] FIG. 2 is a graph comparing the reduction in Salmonella
contamination obtained at market age by injection of eggs with
bacteriophage lysate followed by spraying of chicks.
[0026] FIG. 3 is a graph comparing the reduction in Salmonella
contamination obtained at hatch by injection of eggs with
bacteriophage lysate.
[0027] FIG. 4 is a graph comparing the reduction in Salmonella
contamination obtained at three weeks of age by injection of eggs
with bacteriophage lysate followed by spraying of chicks, in
comparison to egg injection alone or spraying of chicks alone.
[0028] FIG. 5 is a graph comparing the reduction in Salmonella
contamination obtained at market age by injection of eggs with
bacteriophage lysate followed by spraying of chicks, in comparison
to egg injection alone.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0029] Bacterin is defined in medicine as a vaccine composed of
weakened or dead bacteria which will cause the body to create
antibodies or any other adaptive immune response against the
disease normally caused by the bacteria in the vaccine.
[0030] For this invention, phage lysate bacterin is defined as a
composition comprising bacteria and/or fragments of bacteria killed
by lytic bacteriophage which will induce an immune response, either
cellular or humoral. The bacterial components in such a composition
are produced by infection of bacteria by lytic bacteriophage
followed by production of new bacteriophage particles released in a
subsequent lysis of the bacteria in what is termed a lytic burst.
Substantially all of the bacteria in the suspension are killed by
the infection, meaning that at least 90%, preferably 95%, more
preferably 99% of the bacteria in the suspension are killed by the
bacteriophage. Preferably, residual live bacteria are removed by
means such as centrifugation or filtration so as to render the
bacterin bacteriologically sterile, particularly for the bacterial
host organism used to make the bacterin.
[0031] In the earliest days of bacteriophage research, it was
reported that bacteriophage lysates could be effective eliciting
protective immunity against a variety of bacterial strains
(d'Herelle, F, Le Bacteriophage: Son Role dans l'Immunite, Paris,
Masson et Cie, 1921). Many workers of the period believed that
phage lysates were superior to preparations of whole bacteria for
vaccination to prevent disease (Wollman, et al., "Le phenomene
d'Herelle et la reaction de fixation," Comptes Rendues de la
Societe de Biologie, 85:772, 1921; Hauduroy, P., Le bacteriophage
de d'Herelle, Paris, Librarie le Frangois, 1925. pp. 161-168;
Compton, A., "Immunization in experimental plague by subcutaneous
inoculation with bacteriophage. (Comparison of plain and
formaldehyde-treated phage-lysed plague vaccine.)," Journal of
Infectious Disease, 46:152-160, 1930; Le Louet, G M., "The
bacteriophage as an agent of vaccination against the "barbone'
disease," Journal of the American Veterinary Association,
67:713-717, 1925). Despite numerous examples of efficacy, phage
lysates were not superior in every system examined. Perhaps due to
insufficient understanding of the immune system in those days, and
the variability of observed results, study of bacteriophage as an
immunogen was abandoned.
[0032] More recently, Parry et al. showed that live E. coli were
superior to heat-killed organisms at eliciting specific mucosal
immunity (Parry, et al., "Intestinal immune response to E. coli
antigens in the germ-free chicken," Immunology, 32:731-741, 1977).
The reasons were thought to be alterations of the bacteria that led
to their premature degradation in the intestinal tract.
Bacteriophage-mediated lysis does not alter the bacterial cell
through chemical fixation with, for example, aldehyde
cross-linkers, as is done for some attenuated bacterial vaccines.
Neither does it denature macromolecules, which is how heat
treatment kills bacteria. Thus, phage lysates comprise a means of
effectively killing bacteria while minimally altering their
antigenicity. Preferably, phage lysate bacterin contains primarily
bacterial fragments produced by the burst caused by lytic
bacteriophage in conjunction with bacteriophage particles released
by the burst.
[0033] Preparation of bacteriophage lysates which may be used in
the phage lysate bacterin of this invention can be accomplished by
standard procedures, such as those taught in International
Publication Numbers WO01/50866, WO01/50872, WO01/51066, and
60/497,319, the disclosures of each of which are incorporated
herein by reference. Typically, a culture of bacteria of the
serotype for which immunity is desired is grown to an optical
density at 600 nm (OD.sub.600) of 0.1 to 0.3 in a medium suitable
for culture of the bacteria, and the bacterial culture is
inoculated with bacteriophage known to be lytic for the bacteria.
The culture inoculated with phage is incubated under conditions
favoring infection by the phage until substantially all bacteria
are lysed, which typically occurs after 4 to 8 hours of
fermentation, when the OD.sub.600 can vary from 0.1 to 1.45. The
resultant lysate is treated with nucleases to degrade bacterial and
phage nucleic acids and filtered and/or centrifuged to remove
residual whole, live bacteria prior to use as a vaccine. The phage
lysate bacterin used for vaccination may contain various
physiologically acceptable materials, but will preferably be
substantially free of whole, live bacteria and will be sterile by
culture. For oral administration, or for administration in ovo
where the vaccine is taken up orally by the chick embryo, endotoxin
values may range from 10,000 to 200,000 EU/ml as determined by
Limulus amoebocyte lysate assay, and are most preferably greater
than 60,000 EU/ml and less than or equal to 500,000 EU/ml.
[0034] Vaccination using the phage lysate bacterin is typically
performed by injection or oral administration. Injection may be
subcutaneously, intramuscularly, intravenously, intraperitoneally,
intrapleurally, intravesicularly or intrathecally. Oral
administration includes rectal, inhalation, ocular, otic, or nasal
route, as well as by mouth. The phage lysate bacterin may be
administered orally in, for example, mineral. water, optionally
with 2.0 grams of sodium bicarbonate added to reduce stomach
acidity. Dose and duration of therapy will depend on a variety of
factors, including the patient age, patient weight, and tolerance
of the phage lysate bacterin. Typically, the vaccine may be
administered in a single dose or in a series of doses spaced
sequentially to enhance the immune response. The amount of
bacterial and bacteriophage material in each dose may be the same
or the amounts may be varied to promote the boost effect.
Variations in immunization procedure within the skill of the art
are within the contemplation of this invention.
[0035] The phage lysate bacterin of this invention used to
development enhanced protective immune response in animals in need
of protection from bacterial infection, including especially
mammals and birds. Animals which may be immunized by vaccination
with the phage lysate bacterin of this invention include domestic
animals such as horses, cattle, llamas, sheep, goats, rabbits, cats
dogs, chickens, ducks, geese, turkeys, as well as rodents and
primates, including humans.
[0036] The present invention may be used in many animal husbandry
industries. This includes, but is not limited to, the breeding,
raising, storing, and slaughter of chickens, turkeys, ducks, geese,
and other avian species. Where appropriate, the application of a
bacteriophage cocktail is within the contemplation of the present
invention, including a preferred mode where each bacteriophage may
be grown on a separate bacterial host strain, resulting in efficacy
against a broader range of bacterial strains than might be achieved
by use of a single bacteriophage and host strain. In one
embodiment, the working phage concentration may range from
1.times.10.sup.5-1.times.10.sup.10 pfu/ml.
[0037] In one embodiment, the present invention provides an oral
bacterin vaccine to prevent disease caused by Salmonella in poultry
flocks by substantially reducing the percentage of birds colonized
by Salmonella species throughout the growth cycle to market age. In
a preferred embodiments, a Salmonella phage lysate bacterin of
equal proportions of S. enteritidis, S. agoyia, and S. newport is
prepared by fermentation and filtration. The lysate is preferably
sterile with respect to bacteria. The vaccine may be administered
orally by injection into hatchery eggs, for example on Day 18 of
incubation, and/or sprayed onto the feathers-of newly hatched
chicks.
[0038] In another embodiment of the invention, bacterin vaccines
are produced for both typhoidal and non-typhoidal Salmonella
species. In a preferred embodiment of the invention, non-typhoidal
Salmonella species to which bacterin vaccines are produced include
Serogroup B and D strains of Salmonella.
Treatment of Fertilized Eggs
[0039] The invention contemplates the treatment of fertilized avian
eggs by injection of phage lysate bacterin containing at least one
bacteriophage or, more preferably, a cocktail of bacteriophage.
While this embodiment describes the treatment of chickens, the
invention contemplates treatment of chickens, turkeys, ducks,
geese, and other avian species. After the. fertilized eggs are
collected in the Fertilized Egg Collection Site, the fertilized
eggs may be immediately, injected with phage lysate bacterin, or
may be incubated and injected with phage lysate bacterin on any day
up to the day of hatching. Preferably, eggs are injected between
days 17 and 19 of incubation, however this may vary with the breed
and the type of incubator used. Most preferably, eggs are injected
on day 18 of incubation. It is common practice well known to those
skilled in the art to inject fertilized eggs with vaccines to avian
diseases such as Marek's disease during incubation. Phage lysate
bacterin may be injected into fertilized eggs or may be mixed with
vaccines and injected at any point during egg incubation. It has
not been possible to consistently eliminate Salmonella from breeder
flocks, and, consequently, Salmonella may be present in and on the
surface of fertilized eggs; conditions in incubators promote
multiplication of the organism, and chicks may become infected as
they pip out of the egg. Aggressive washing of eggs and the use of
disinfectants of sufficient strength to eliminate all bacterial
contamination is not desirable with fertilized eggs. In this
setting, injecting phage lysate bacterin directly into the eggs may
provide an improved means of immunizing hatched chicks, poults,
ducklings, goslings, or young of other avian species.
Treatment of Newly Hatched Chicks and Older Birds
[0040] After the birds are hatched, the birds may be sprayed with
phage lysate bacterin before they are transferred to a chicken
house or to a farm. Immediately after hatching, chicks are sprayed
with various viral vaccines (Newcastle, bronchitis, INDIA) which
are ingested as the animals preen their feathers. A small
percentage of chicks are Salmonella-positive at this point in time
(see comments above about Salmonella on eggs); however, once
introduced into chicken houses, contamination spreads rapidly to
all animals in the house. Application of phage lysate bacterin
immediately after hatching and before transfer to chicken houses
may reduce the risk of the bacterium being spread between chicks
during placement and growout. During raising in the chicken house
or farm, the birds may be provided with phage lysate bacterin in
their drinking water, food, or both.
Delivery of Bacteriophage to Avian Eggs
[0041] This invention contemplates the delivery of phage lysate
bacterin to eggs by injection. One traditional method of injecting
eggs is injection by hand. Although skilled operators can inject
eggs by hand with some success, the speed and accuracy of the
process is limited. Additionally, hand injection of eggs, even by
skilled operators, cannot always guarantee the continuous repeated
precision delivery of materials to a desired particular location
within each egg. As an alternative, active substances may be
injected into eggs by automated machinery such as the Inovoject
system (Embrex, Inc., Research Triangle Park, N.C.).
[0042] Similarly, an alternative technique for treating poultry to
obtain desired results has been hand injection of very
young--typically day-old--chicks. As in the hand injection of eggs,
speed and precision are limited. Furthermore, injection so soon
after hatch places significant stress on the young chicks.
[0043] Depending upon the purpose for which the egg is being
treated, the location of injection will vary. For certain purposes,
the substance to be injected into the egg needs to be delivered to
the amniotic fluid near the small end of the egg, for other
purposes the material needs to be delivered to the air sac end of
the egg, and there may even arise occasions when a substance should
be delivered to the embryo itself. Nevertheless, where eggs are
being incubated to produce live poultry, care must be taken to
avoid injuring the embryos during the injection and delivery of
fluid substances. Individual eggs, however, can vary widely in size
with accompanying associated differences in the distance between
the shell and the location to which delivery of a fluid substance
is desired. These differences can complicate the task of
consistently supplying a desired substance to a particular location
within each of a large number of eggs at a fast rate of speed.
[0044] As would be expected, when an injection device is placed in
contact with a large number of eggs--which indeed is the purpose of
such a machine--contamination of any one or more of the needles may
occur. For example, a needle encountering an egg which has died
during incubation can easily become contaminated by the materials
in the dead egg.
[0045] As another consideration in egg injection, where specific
vaccines or other sensitive materials are to be delivered, the
specific quantity delivered is often an important parameter. This
is especially true when very small quantities of materials must be
delivered. For example, in some treatments of avian embryos,
microliter quantities are often desired. The large systems of pumps
and tubing used heretofore in injection machines makes accurate and
precise delivery of such small quantities rather difficult.
[0046] Several injection devices seal the injection hole after
injection to prevent leakage and contamination. U.S. Pat. No.
4,593,646 to Miller et al. discloses a method and apparatus for
automatic egg injection in which support plates hold and properly
position a plurality of injection devices and eggs. Each egg is
sealed after injection by heat coagulating the albumin located near
the injection hole. An additional sealant is then applied to the
outer shell by dipping each egg into a bath of the sealant. The
'646 patent does not disclose sealing the egg prior to
injection.
[0047] U.S. Pat. No. 4,040,388 to Miller discloses a method and
apparatus for automatic egg injection in which the downwardly
facing small end of an egg is punctured. The portion of the device
which punctures the egg is heated in the '388 method, allegedly
sterilizing the exterior of the egg (thus preventing infection
during injection) and also sealing the hole by heat coagulating a
small portion of the egg albumin. The '388 patent does not disclose
sealing the egg prior to injection.
[0048] U.S. Pat. No. 2,477,752 to Kiss discloses a method of
injecting fertile eggs for the purpose of producing chicks having
down of predetermined colors. The '752 patent discloses injecting
the egg manually with a syringe and thereafter by sealing the
opening in the egg. While the patent states that care should be
taken to prevent air from entering the egg, no method for
preventing the entrance of air is provided. Sealing the egg prior
to injection is not disclosed.
[0049] U.S. Pat. No. 5,136,979 to Paul et al. discloses an
apparatus and method for injecting a plurality of eggs to the same
depth and location even when the eggs are of varying sizes and are
misaligned. The apparatus includes a means for sterilizing the egg
punch and needle sections after each injection.
[0050] U.S. Pat. No. 5,056,464 to Lewis discloses an apparatus and
method for injecting a plurality of eggs in which a suction cup
apparatus is used for grasping each egg.
[0051] U.S. Pat. No. 4,903,635 to Hebrank discloses a high speed
automated injection system in which eggs are lifted using suction
devices and separate devices are used for forming an opening in the
egg shell and for injecting a fluid substance.
[0052] Some egg injection devices deliver material through the
small end of an egg into the albumin. Injecting material through
the large end of an egg and into the air sac above the albumin is
not appropriate for delivery of all materials. Delivery into the
albumin, however, increases the risk of leakage of albumin and
ingress of air and contaminants after injection. Methods of
injecting material into the albumin of eggs on a rapid basis should
preferably provide means for preventing air and contaminants from
entering the albumin, and means for preventing leakage of albumin,
after injection.
[0053] Conventionally, the physical injection has been typically
targeted at preferred positions within the egg in order to
administer the substance into specific developing regions of the
embryo. As understood by those of skill in the art, as the
incubation period progresses towards maturity (i.e., hatching), the
embryo and its membranes, e.g., the air cell, the allantois, and
yolk sac, correspondingly change in both volume and position within
the egg shell. Additionally, the quantitative volume of the
enclosed fluids vary as well; for example, the density of the
allantois (fluid, solid) varies as a function of time over the
incubation period.
[0054] Thus, selection of both the site and time of treatment can
impact the effectiveness of the injected substance as well as the
mortality rate of the treated embryos. See e.g., U.S. Pat. No.
4,458,630 to Sharma et al., U.S. Pat. No. 4,681,063 to Hebrank, and
U.S. Pat. No. 5,158,038 to Sheeks et al.
[0055] In one preferred embodiment, this invention contemplates the
use of an automated egg injector. An example of such an injector is
the Embrex Inovoject.RTM. system.
[0056] In a preferred embodiment, pathogens will be a member of the
genus Salmonella. In a most preferable embodiment, pathogens will
include S. enteriditis, S. heidelberg, S. kentuckii, S. hadar, and
S. typhimurium.
Bacteriophage Cocktails
[0057] This invention also contemplates phage lysate bacterins
generated using bacteriophage cocktails, which may be custom
tailored to the pathogens that are prevalent in a certain
situation. Typically, pathogenic bacteria would be initially
isolated from a particular source (e.g., a contaminated flock or
location such as a henhouse) and susceptibility testing of the
pathogens to various bacteriophage strains would be performed,
analogous to antimicrobial susceptibility testing. Once each
pathogen's phage susceptibility profile is determined, the
appropriate phage cocktail can be formulated from phage strains to
which the pathogens are susceptible and administered to the patient
or applied in the environment. Since phage cocktails will typically
be constructed of phage active against avian and human food-borne
pathogens, isolation of such phage would be most successful from
the waste of poultry farms and poultry processing plants.
Typically, the phage cocktail will include one or more
bacteriophage according to this invention. Thus, it may be
appropriate to use certain phage cocktails in agricultural settings
where there are certain human pathogens such as Salmonella and
Campylobacter inherent to poultry or livestock and which
contaminate the environment of such animals on an ongoing basis,
resulting in a continuing source of infection by such
pathogens.
[0058] Bacteriophage cocktails may be applied by contemporaneous
administration of the various phage--that is, they may be applied
at the same time (e.g., in the same application), or may be applied
in separate applications spaced in time such that they are
effective at the same time.
[0059] Other bacteria that may be lysed by phage to produce
bacterin within the contemplation of the present invention include,
inter alia, Campylobacter, E. coli O157:H7, E. coli K88 and F18
serotypes, Acinetobacter baumanii, Actinobacillus pleuropneumoniae,
Aeronoias hydrophila, Brucella abortus, Campylobacter jejuni,
Clostridium perfringens, Corynebacterium bovis, Klebsiella
pneumoniae, Lactococcus garvieae, Listeria species such as Listeria
monocytogenes and Listeria ivanovii, Pasteurella multocida,
Pasteurella haemolytica, Pseudomonas aeruginosa, Pseudomonas
plecoglossicida, Shigella species, Staphylococcus aureus,
Streptococcus equi, Vibrio aziguillaruni, Vibrio vulnificus,
Yersinia enterocolitica, and Yersinia pseudotuberculosis. Other
bacteria within the contemplation of the invention, such as for
example those set forth supra, are cultured using media and methods
known to those of skill in the art. These bacteria are lysed by
adding bacteriophage to the culture medium following the methods
described herein, and the phage concentration is optimized to
target lysis which produces the maximum antigen yield. Lytic phage
specific for these bacteria may be isolated by methods analogous to
those described in Int. Pat. Pub. WO 01/50866, herein incorporated
by reference.
[0060] The invention will now be described in more detail in the
following non-limiting examples with reference to the drawings. The
examples are for illustration only and do not limit the scope of
the present invention in any way, which is defined only by the
claims. All references cited are hereby incorporated by reference
herein in their entirety. While the invention has been described
with reference to specific embodiments thereof, it will be
appreciated that numerous variations, modifications, and
embodiments are possible, and accordingly, all such variations,
modifications, and embodiments are to be regarded as being within
the spirit and scope of the invention.
EXAMPLES
Example 1
Preparation of Bacteriophage Lysate Bacterin
[0061] A phage lysate of three Salmonella serotypes (5 strains) is
produced by independent lysis of the bacteria by six clonal
bacteriophages, or monophages. Salmonella strains were obtained
from a variety of environmental sources such as poultry production
and processing facilities. Naturally occurring bacteriophages were
isolated from various U.S. environmental sources, including waters
of Chesapeake Bay and used without further modification. Each of
the monophages was isolated using standard techniques of mixing
serial dilutions of the putative phage source with top agar that
was then plated over a lawn of Salmonella species contained in
bottom agar. The plates were incubated overnight and inspected for
areas of bacterial lysis, or plaques. Plaques were isolated,
eluted, and re-plated serially until clonal as determined by
several features including a stable restriction digest pattern,
stable and homogeneous morphology by electron microscopy, and
stable host range. and lytic titer against host Salmonella strains.
The particular bacteriophages are described in more detail in U.S.
Provisional Patent Application No. 60/497,319, incorporated herein
by reference. Bacteriophages are lytic tailed double-stranded DNA
phages that either belong to the Siphoviridae family as classified
by Ackermann and Berthiaume (1995, "Atlas of virus diagrams," CRC
Press, Boca Raton, Fla.), or are unclassified isometric DNA
phages.
[0062] Salmonella phage lysate containing 3 Salmonella serotypes
comprising 4 strains may be prepared as follows. A total of six
monophages may be used to prepare the lysates. The Salmonella
strains and phage used to lyse them are indicated as follows:
TABLE-US-00001 Salmonella Strains Bacteriophage S. Enteritidis 378
SPT-1 S. Enteritidis 236 SIT-128, SDT-15 S. Newport 388 SBA-178,
SBA-1781 S. Agona 121 SSE-121
[0063] The isolated Salmonella bacteriophage SPT-1, SBA-178,
SBA-1781, SIT-128, SSE-121 and SDT-15 described above were
deposited on Jun. 24, 2003 with the ATCC and received ATCC Deposit
Accession Nos. PTA-5281, PTA-5284, PTA-5282, PTA-5285, PTA-5283 and
PTA-5280, respectively. Separate Salmonella phage lysates are
prepared as follows using each monophage listed in the table. These
are blended in equal proportion.
[0064] Working phage stocks were concentrated clonal bacteriophage
maintained in phosphate-buffered saline at 4.degree. C. in sealed
glass ampoules sealed with a rubber septum and retaining ring, or
as preparations prepared at the same concentration in 5% calcium
gluconate and subsequently freeze-dried and stored at room
temperature. Freeze-dried preparations are suspended in
pyrogen-free water or sterile phosphate-buffered saline immediately
before use. Individual disposable vials of bacteriophage seeds are
diluted to 10.sup.8 pfu/ml in water, phosphate-buffered saline, or
Luria broth.
[0065] Frozen bacterial seeds are rapidly thawed immediately prior
to inoculation. Bacteria are prepared as an overnight culture in LB
of the desired strain of Salmonella. Bacteria subculture is
prepared by inoculating the contents of a 0.5 ml vial of the
bacteria into a flask containing 70 ml of bacterial growth medium.
A working seed subculture is prepared by inoculating the working
seed into a flask containing an appropriate volume of bacterial
growth medium. The inoculum constitutes approximately 1 % of the
total fermenter volume. Working seed bacterial cultures are
incubated on a shaker at 150.+-.10 rpm for 1 day at 37.degree.
C..+-.2.degree. C.
[0066] Production seed culture is prepared by inoculating bacterial
working seed subculture into a 10 L fermenter containing the
bacterial growth medium. The inoculum constitutes approximately 5%
of the total production fermenter volume. A pre-determined optimal
volume of bacteriophage seed stock representing one of the
monophages is added along with the inoculum. The appropriate
bacteriophage seed stock(s) for the given bacterial host is added
along with the inoculum. Bacteriophage are added at a predetermined
multiplicity of infection (MOI) as established for each bacterial
host-bacteriophage pair. Fermentation is carried out at 37.degree.
C. with periodic or continuous monitoring of the OD600 until
optimal lysis for each host-bacteriophage pair has occurred.
[0067] Bacterial cell suspensions containing phage are cleared of
live bacteria and large bacterial fragments by either low speed
centrifugation, or by tangential flow filtration. Supernatant
fluids containing Salmonella antigens and bacteriophage are then
filtered through an inert 0.22 .mu.m pore size filter to remove
intact bacteria. All filtrates are then treated with DNAse I and
RNAse A. Following nuclease digestion, the Salmonella antigens and
bacteriophage are collected, washed, concentrated, and exchanged
into phosphate-buffered saline by tangential flow filtration. The
tangential flow filtration process removes medium components,
digested nucleic acids, and the nucleases. The resulting product is
then filtered through an inert 0.22 .mu.m filter and handled
aseptically.
Example 2
Injection of Chicken Eggs and Treatment of Chicks
[0068] The objective of this example was to determine the effects
of injecting eggs and treating chicks in a commercial setting using
a bacteriophage preparation specific for Salmonella. Salmonella
species are a type of pathogenic Gram negative bacterium often
found in the intestines of animals (including humans) and in the
environments in which food producing animals are raised and
processed (e.g., in soil, water, vegetation and on the surfaces of
equipment, floors, and walls). The major pathogenic species
include: S. typhimurium, S. enteritidis, S. leidelberg, S. newport,
S. hadar, S. kentuckii, andS. thomson. Salmonella can cause
salmonellosis, a serious and sometimes fatal illness. For a
thorough discussion of Salmonella and the risks to human health
posed by the pathogen, see US FDA CFSAN, Foodborne Pathogenic
Microorganisms and Natural Toxins Handbook, Jan. 1996 with updates
(available at www.cfsan.fda.gov/.about.mow/chap1.html), the
CDC/Foodnet Salmonella surveillance system and FSIS/USDA data at
http://www.foodsafety.gov/ and http:H/www.cdc. gov/foodnet/.
[0069] Bacteriophage employed in this experiment consisted of a
cocktail of five bacteriophage directed against pathogenic
Salmonella species. Specifically, the preparation consists of five
clonal bacteriophage targeting S. typhimurium, S. enteritidis, S.
heidelberg, S. newport, S. hadar, and S. kentuckii. To conduct this
experiment, two egg hatchers were randomly paired. At the time of
transfer of fertilized eggs to the hatcher, eggs from one hatcher
were processed as usual in the commercial setting, which included
injection of eggs with vaccine for Marek's disease. The eggs from
the other hatcher were injected with phage specific for Salmonella
mixed in with the Marek's vaccine. 200 ml of a bacteriophage
cocktail at 10.sup.10 pfu/nil phage were introduced into a 1600 ml
vaccine bag after prior removal of 200 ml of diluent to accommodate
the phage. Injection of 0.1 ml of the phage/vaccine mixture per egg
thus resulted in injection of a dose of 1.25.times.10.sup.8
pfu/egg.
[0070] At the time of hatching, chicks from the control hatcher
receiving Marek's vaccine but no phage were processed and moved
first. 40 chicks were collected prior to spray vaccination and
placed in labeled egg boxes for sampling for Salmonella. The
remaining 960 chicks were sprayed with 7.0 ml of
Newcastle/Bronchitis vaccine per 100 chicks, then taken to the farm
for placement. The farm chicks remained in the chick boxes until
the phage-treated chicks were delivered to the farm so that they
were placed near water and food at approximately the same time.
[0071] Chicks from phage-treated eggs received a second application
of phage at chick processing with spray vaccines. Again, prior to
spray vaccination, 40 chicks were collected and placed in labeled
egg boxes for sampling for Salmonella. Each box of 100 chicks was
sprayed with 7.0 ml of Newcastle/Bronchitis vaccine containing
bacteriophage at concentration of 2.5.times.10.sup.9 pfu/ml. This
resulted in a dose of 1.7.times.10.sup.6 pfu/chick.
[0072] Chicks housed at the farm were arrayed so that no phage
would be tracked into the chambers housing the control birds.
Disinfectant foot pans were used and maintained at each closed
chamber door. The traffic flow path was designed to isolate the
phage. 960 chicks from each hatcher were placed into the trial
house (1960 birds total), with 60 birds per pen.
[0073] Chicks were sampled periodically to determine the rate of
contamination with Salmonella. For sampling of the newly hatched
birds collected prior to spray vaccination, the gastrointestinal
tracts were-collected, diluted in buffered peptone water, held at
room temperature for 48 h, then qualitatively assayed for
Salmonella using tetrathionate enrichment and supplemental
polymerase chain reaction amplification using the BAXO PCR test kit
(DuPont Qualicon, Inc., Wilmington, Del.) according to the
manufacturer's instructions.
[0074] A second group was sampled at three weeks of age. Ceca were
collected from 10 birds/pen, for a total n=320. Specimen collection
occurred over two days. Chick weights were recorded prior to
collecting the ceca. The ceca were assayed qualitatively for the
incidence of Salmonella, using tetrathionate enrichment and
supplemental polymerase chain reaction amplification using the
BAX.RTM. PCR test kit (DuPont Qualicon, Inc., Wilmington, Del.)
according to the manufacturer's instructions. Any positives from
the phage-treated group were isolated for phage sensitivity
testing.
[0075] A third group was sampled at market age. Ceca were collected
from 10 birds/pen, for a total n=160. Chick weights were recorded
prior to collecting the ceca. The ceca were assayed qualitatively
for the incidence of Salmonella, using tetrathionate enrichment and
supplemental polymerase chain reaction amplification using the
BAX.RTM. PCR test kit (DuPont Qualicon, Inc., Wilmington, Del.)
according to the manufacturer's instructions. Any positives from
the phage-treated. group were isolated for phage sensitivity
testing. This sampling was done at a market age of 39-40 days.
Market age can vary between 42-60 days for broilers, depending on
the target weight of the bird, and can be longer for roasters.
[0076] The results of this experiment are illustrated in FIGS. 1
& 2 with supporting data shown in Table 1 & 2. The data
demonstrate that phage treatment produced a statistically
significant reduction in the incidence of Salmonella in phage
treated birds. Testing at 3 weeks of age showed that only 0.6% of
phage-treated birds were Salmonella positive, as compared to 11% of
control birds. At market age, 1% of phage-treated birds were
Salmonella positive, as compared to 16%. The results indicate that
application of phage in the egg combined with spray treatment of
chicks is a useful intervention for the reduction of Salmonella
during poultry production.
Example 3
Injection of Eggs
[0077] The objective of this example was to compare the levels of
Salmonella reduction obtained by first intentionally contaminating
eggs with Salmonella, then injecting eggs with a bacteriophage
preparation specific for Salmonella to the levels obtained by
spraying chicks, and to compare each group to a group treated both
by injection of eggs and spraying of chicks.
[0078] Bacteriophage employed in this experiment consisted of a
cocktail of five bacteriophage directed against pathogenic
Salmonella species. Specifically, the preparation consists of five
clonal bacteriophage targeting S. typhimurium, S. enteritidis, S.
heidelberg, S. newport, S. hadar, S. kentuckii, and S. thontson. To
conduct this experiment, eggs were collected from the hatchery. 40
of these eggs were randomly collected and sampled for natural
Salmonella contamination by crushing the whole egg into 50 ml
buffered peptone water and assaying for Salmonella as described in
Example 1 above. 440 eggs were contaminated with 10.sup.7 cfu of a
mixture of S. Kentuckii, S. heidelburg, S. hadar, and S.
typhimurium. For contamination, the eggs were allowed to come to
room temperature for 2 h. The Salmonella inoculum was prepared by
combining equal aliquots from individual, overnight cultures of
each strain. Each egg was inoculated with 20 .mu.l (2.times.10
.mu.l loops) of the Salmonella cocktail at room temperature. The
cocktail suspension was spread over the blunt (air cell) end of the
egg and allowed to dry. Following inoculation, the eggs were
incubated overnight, then ten eggs were sampled as above to
determine the level of Salmonella contamination by preparing a
shell rinse sample in 50 ml buffered peptone water, an
eggshell/membrane sample in 10 ml buffered peptone water, and an
egg contents sample in 50 ml buffered peptone water. Each rinse and
eggshell sample was serially diluted out to 10.sup.-5 or to
10.sup.-4 respectively from the initial pre-enrichment, using
buffered peptone water, and all dilutions were incubated for
plating. The egg contents were diluted to 10.sup.-2 in buffered
peptone water and incubated for plating. All samples and dilutions
will be incubated at 37.degree. C. for 24 hours. A 10 .mu.l aliquot
of each sample was then plated and be incubated overnight. The
extinction point from each dilution series of Salmonella will be
recorded to estimate the log number of Salmonella in the original
sample.
[0079] On day 18 of incubation, 50 ml of a bacteriophage cocktail
at 5.times.10.sup.10 pfu/ml phage were introduced into a 400 ml
Marek's vaccine bag. Injection of 0.1 ml of the phage/vaccine
mixture per egg thus resulted in injection of a dose of
5.6.times.10.sup.8 pfu/egg. The phage preparation was identical to
that described in Example 1 above. For control eggs, 50 ml of
phosphate-buffered saline was introduced into an identical 400 ml
Marek's vaccine bag. One group of 220 eggs was manually injected
with 0.1 ml of the control preparation, while a second group of 220
eggs was injected with 0.1 ml of the phage preparation and the eggs
incubated until hatching.
[0080] On the day of hatch, gastrointestinal tracts, yolk sacs, and
whole bird feather rinses were collected from 20 chicks from each
group. Each sample was assayed qualitatively for the presence of
Salmonella, and 10 gastrointestinal samples and 10 feather rinse
samples from each group were also quantitatively assayed by serial
dilution, and plating as above.
[0081] After day-of-hatch sample collection, groups of 80 chicks
were sprayed with either 28 ml of a phage solution at
5.times.10.sup.9 pfu/ml, or 28 ml of a control solution. At three
weeks of age and at market age, ceca and feather rinses were
sampled for Salmonella using both quantitative and qualitative
assays as described above.
[0082] Sampling of the inoculated eggs at 24 hours documented that
100% of eggs were contaminated with Salmonella recoverable from
whole egg washes, shell and membrane fractions, and from the egg
contents. FIG. 3 shows that phage treatment produces a
statistically significant reduction of intentional Salmonella
contamination in the gastrointestinal tracts but not the feathers
of birds sampled at hatch. Persistent feather contamination, likely
acquired during hatching from egg shells, since the shells were not
previously sprayed with phage, then leads to gastrointestinal
contamination of chicks from eggs injected with phage.
Substantially less colonization occured in birds from injected eggs
that were subsequently sprayed, as seen in FIGS. 4 and 5, which
show the birds at 3 weeks and at market age. The principal benefit
is from injection, since spraying of birds alone has little effect,
as seen in FIG. 4.
[0083] For purposes of charity of understanding, the foregoing
invention has been described in some detail by way of illustration
and example in conjunction with specific embodiments, although
other aspects, advantages and modifications will be apparent to
those skilled in the art to which the invention pertains. The
foregoing description and examples are intended to illustrate, but
not limit the scope of the invention. Modifications of the
above-described modes for carrying out the invention that are
apparent to persons of skill in medicine, bacteriology, immunology,
infectious diseases, pharmacology, and/or related fields are
intended to be within the scope of the invention, which is limited
only by the appended claims.
[0084] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All these publications
and patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
* * * * *
References