Note: Descriptions are shown in the official language in which they were submitted.
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BOVINE FOOTROT TREATMENT AND PREVENTION
FIELD OF THE INVENTION
The present invention relates to compositions useful for the treatment and
prevention of footrot and, in particular, bovine footrot.
BACKGROUND OF THE INVENTION
Acute bovine footrot, also known as interdigital phlegmon or acute
interdigital
phlegmon (AIP), is a common infection in cattle [ 1,11 ] . The actual
prevalence of this
disease in many types of cattle is not fully described; however, in some years
the
prevalence in feedlot animals can reach 10-25 % if preventative measures such
as
feeding antibiotics are not widely implemented.
It is an anaerobic bacterial infection characterized by acute inflammation
which
is manifested as tissue edema and local infiltration of subcutaneous tissues
with
polymorphonuclear granulocytic neutrophils (PMN) [16]. Typically this disease
involves necrosis of the interdigital epidermis and the underlying dermis.
Very often
there is an ascending cellulitis which can result in severe swelling from the
coronet to
the fetlock joint. This infection, if left untreated, can result in sequelae
such as septic
joint involvement which can lead to euthanasia [2].
The infection is thought to be caused by a synergistic association of
anaerobic
bacteria including Bacteroides melaninogenicus, Fusobacterium necrophorum
[12], and
possibly other bacteria such as Dichelobacter (Bacteroides) nodosus [13] or
Actinomyces pyogenes [14]. Recently B.melaninogenicus has been divided into
several
distinct species of bacteria including Porphyromonas sp and Prevotella sp [4].
Fusobacterium necrophorum and B. melaninogenicus have been previously used
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together to experimentally infect cattle [12]. That investigation did not
examine
microscopic pathology or the minimum inhibitory concentrations [MIC's) and
minimum
bactericidal concentrations [MBC's] of the antibiotics) for the pathogens used
in the
experimental infection.
Several treatments have been advocated for acute bovine footrot including
penicillin [11J, oxytetracycline [15), cephalosporins [16], and sulfonamides
[11].
Parenteral antibiotic treatment of individual clinically affected animals is
effective for treating cattle that can be easily handled and frequently
observed. Therapy
in fat cattle (i.e., animals nearly ready for marketing as beef) is
complicated by the fact
that many of these drugs cannot be used without delaying slaughter.
Antibiotics
without withdrawal periods are available for these animals; however, the
expense
remains significant. Currently, recommended therapy for these animals can
involve
daily treatment for up to five days or longer. Cephalosporin, a ~3-lactam
antibiotic, is
one recommended therapeutic regimen. For a 500 kg steer, cephalosporin therapy
for
five days can approach $50 in antibiotic cost alone for a single episode of
the disease.
This clearly does not include the costs of manpower for giving the treatments,
the costs
of lost production (e.g., reduced weight gain) in affected cattle, or the
significant
animal suffering that occurs as a result of this infectious lameness.
There are potentially devastating effects of this disease in breeding bulls if
the
infection occurs during breeding season and libido is reduced. Conventional
antibiotic
therapy in mature breeding bulls is problematic because of the frequency of
treatments
and the massive volume of antibiotic required, in addition to the cost of
antibiotics.
Although infection can be minimized in some types of cattle through feeding
antibiotics, fat cattle are again at high risk because of our inability to use
these drugs
due to withdrawal times. Preventative measures, such as footbaths, are
recommended
in many parts of the world but are not practical under many circumstances. The
potential environmental implications of using compounds such as blue stone and
formaldehyde are also a consideration.
Animals affected with AIP are believed to develop immunity, but the importance
of this immunity is unclear [2]. In many acute infectious inflammatory
diseases
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phagocytosis by polymorphonuclear granulocytic neutrophils (PMN) is a central
mechanism in the resolution of infection, but these cells have never been
specifically
evaluated in the context of acute bovine footrot. Specific immunity to
etiologic agents
of footrot also may be important in resolution of the infection [2]; however,
studies
have yet to be conducted on precisely how these cells are involved in the
mechanisms
of this process.
The development of a therapeutic agent and/or a vaccine, additional tools for
cattlemen to minimize the effects of footrot, would be worthwhile and a
significant
contribution to sustainable agricultural practices.
Traditionally, Fusobacterium necrophorum has been described as the cause of
bovine footrot [2-4]. Vaccines based on this microorganism are known and/or
available
[5,8,9], but efficacy is questionable and use is not broad. Use of
6-substituted 3-nitroimidazo[1,2,b]pyridazine for the control of footrot and
liver lesions
caused by Fusobacterium necrophorum has also been disclosed [ 10] .
SUMMARY OF THE INVENTION
This invention provides a method of preventing or treating bovine footrot by
administering vaccine compositions comprising at least one of the causative
agents of
bovine footrot, i.e., Porphyromorzas and Prevotella and/or subunits and/or
toxins
thereof. Compositions comprising Porphyromonas levii and Prevotella intermedia
are
preferred. Protectively immunogenic vaccine compositions may also contain a
specific
bovine immunoglobulin GZ (IgG2) destroying toxin of Porphyromonas levii and a
~3-
lactamase enzyme of Prevotella intermedia, either or both of which can be
inactivated
and included in the vaccine composition. The invention also provides a method
of
treating or preventing bovine footrot by administering compositions comprising
at least
one neutralizing agent, e.g., an antibody, to a causative agent of bovine
footrot, i.e.,
Porphyromonas and Prevotella and/or subunits and/or toxins thereof.
Compositions
comprising antibodies to Porphyromonas levii and Prevotella intermedia and/or
their
toxins) or subunit(s) are preferred. Effective compositions may contain
antibodies to
immunoglobulin proteases, including a specific bovine immunoglobulin G, (IgG2)
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destroying toxin of Porphyromonas levii and/or antibodies to antibiotic
resistance
enzymes, including a (3-lactamase enzyme of Prevotella intermedia.
Our isolation techniques and our immunology studies suggest that
Porphyromonas (and especially P. levii) and Prevotella (and especially P.
intermedia)
are more appropriate vaccine candidates than Fusobacterium necrophorum. Using
surgical biopsy techniques and stringent anaerobic culture methodology, we
have not
isolated Fusobacterium necrophorum from internal infected tissues in a single
case of
bovine footrot.
The invention provides compositions and methods for preventing or treating
infectious lameness, particularly in cattle, using the bacteria Porphyromonas
and/or
Prevotella, toxins isolated from these bacteria, and neutralizing agents which
may be
used in the compositions. The invention provides methods of preparing and
methods of
using a bacterial protease toxin, an antibiotic destroying toxin, and
antibodies to the
toxins.
Accordingly, in one aspect, the invention provides compositions for preventing
or treating footrot comprising Porphyromonas and/or Prevotella. Compositions
comprising subunit(s) and/or toxins) of these bacteria are also provided, as
are
methods of preparing these compositions.
A further aspect of the invention is a method of preventing or treating
infection
in an animal comprising administering to the animal an effective amount of a
composition comprising Porphyromonas and/or Prevotella and/or subunit(s)
and/or
toxins) thereof.
Another aspect of the invention provides isolated toxins from Porphyromonas
and/or Prevotella. Methods of preparing the toxins are also provided, as are
antibodies
to the toxins and a method of passive immunization using the antibodies.
Yet another aspect of the invention provides an experimental model useful for
evaluating the effectiveness of possible preventatives or cures for bovine
footrot
comprising a bovine which has been administered Porphyromonas or Prevotella,
especially in conjunction with Fusobacterium necrophorum.
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In yet a further aspect, the invention provides compositions for preventing or
treating footrot comprising at least one agent which neutralizes Porphyromonas
and/or
Prevotella and/or subunit(s) and/or toxins) thereof.
In a still further aspect, the invention provides methods for treating or
preventing footrot comprising administering to an animal suffering from or
susceptible
to footrot an effective amount of a composition which enhances neutrophil-
mediated
resolution of infection by bacteria associated with footrot.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. lA through 1D are electrophoretic transfers of biotin labeled
immunoglobulins that have been incubated with IgGz destroying toxin-containing
extract from Porphyromonas levii. In Fig. lA IgG was used as the
imrnunoglobulin; in
Fig. 1B IgM was used; in Fig. 1C IgG, was used; and in Fig. 1D IgG2 was used.
Note
that fragments of immunoglobulins are present in Figs. lA and 1D, but not in
Figs. 1B
and 1C, showing that this protease is IgG2 specific.
Fig. 2 is a photograph illustrating the cefinase method of detecting (3-
lactamase
enzyme. The upper disc is a disc exposed to Prevotella intermedia, the lower
left disc
a negative control, and the lower right disc (marked (3) is exposed to
commercially
purified (3-lactamase enzyme-positive control. The results show that
Prevotella
intermedia produces a ~i-lactamase enzyme.
Fig. 3 is a silver stain SDS-polyacrylamide gel of varying concentrations of
protein from a 40-50% ammonium sulfate precipitation of extracts from
Prevotella
intermedia. These extracts retained potent ~i-lactamase activity as assessed
by the
cefinase and other assays.
Figs. 4A through 4C are Western immunoblots of the reaction of bacterial
antigens of Fusobacterium necrophorum (Fig. 4A), Prevotella intermedia (Fig.
4B),
and Porphyromonas levii (Fig. 4C) with serum collected from cattle
experimentally
infected with footrot. The results show that animals developed immunity, as
evidenced
by the development of serum antibodies to Prevotella intermedia and
Porphyromonas
levii, but not to Fusobacterium necrophorum, after experimental infection.
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Figs. SA through SD are Western immunoblots of the reaction of bacterial
antigens from Fusobacterium necrophorum (FN), Prevotella intermedia (5.2), and
Porphyromonas levii (7.5) with acute serum - taken the day clinical signs were
noticed
(A); and convalescent serum - taken 28 days after appearance of clinical signs
(C) from
four animals (Figs. SA through SD) naturally infected with bovine footrot.
Note the
generally minimal response to Fusobacterium necrophorum and the increasing
intensity
of banding patterns to either Prevotella iraermedia or Porphyromonas levii.
Fig. 6A is a graphical representation of the footrot scores in Group 1 animals
(infected with Porphyromonas levii and Fusobacterium necrophorum). Data are
mean
scores ~ standard errors. The arrow indicates the time of treatment and the
asterisks
represent a statistically significant (P < 0.05} reduction in footrot score
compared to
pre-treatment score.
Fig. 6B is a graphical representation of the foouot scores in Group 2 animals
(infected with Prevotella intermedia and Fusobacterium necrophorum). Data are
mean
scores ~ standard errors. The arrow indicates the time of treatment and the
asterisks
represent a statistically significant (P < 0.05) reduction in footrot score
compared to
pre-treatment score.
Fig. 7 is a graphical representation of the footrot scores of animals
(combined
data of Group 1 and Group 2). Data are mean scores ~ standard errors. The
arrow
indicates the time of treatment and the asterisks represent a statistically
significant
(P < 0.05) reduction in footrot score compared to pre-treatment score.
Fig. 8 is a graphical representation of the footrot scores of animals
(combined
data of Group 1 and Group 2 - excluding values from a non-responding animal).
Data
are mean scores ~ standard errors. The arrow indicates the time of treatment
and the
asterisk represents statistical significance compared to pre-treatment score
(P < 0.05).
Fig. 9A is a nitrocellulose transfer of biotinylated IgG exposed to culture
supernatant of P. levii. From left to right are increasing times (hours) of
exposure.
Lane A = 0, Lane B = 0.5, Lane C = 1, Lane D = 2, Lane E = 4, Lane F = 8,
Lane G = 24, Lane H = 48, and Lane I = 72h time samples, showing increasing
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intensity of the degradation bands smaller than the heavy and light chains as
incubation
time increases and the near complete degradation of the heavy chain by 24h.
Fig. 9B is a nitrocellulose transfer of biotinylated IgM exposed to culture
supernatant of P. levii. From left to right are increasing times (hours) of
exposure.
Lane A = 0, Lane B = 0.5, Lane C = 1, Lane D = 4, Lane E = 8, Lane F = 24,
Lane G = 48, and Lane H = 72h time samples, showing the absence of degradation
bands, with only the light and heavy chains of IgM evident.
Fig. l0A is a nitrocellulose transfer of biotinylated IgGI exposed to culture
supernatant of P. levii. From left to right are increasing times (hours) of
exposure.
Lane A = 0, Lane B = 0.5, Lane C = l, Lane D = 2, Lane E = 4, Lane F = 8,
Lane G = 24, Lane H = 48, and Lane I = 72h time samples, showing the absence
of
degradation bands, with only the light and heavy chains of IgG, being evident.
Fig. 10B is a nitrocellulose transfer of biotinylated IgG2 exposed to culture
supernatant of P. levii. From left to right are increasing times (hours) of
exposure.
Lane A = 0, Lane B = 0.5, Lane C = l, Lane D = 2, Lane E = 4, Lane F = 8,
Lane G = 24, Lane H = 48, and Lane I = 72h time samples, showing the presence
of
additional bands by 0.5 h, as well as the heavy and light chains of IgGI.
DETAILED DESCRIPTION OF THE INVENTION
The invention is drawn in part to the unexpected discovery that the bacteria
Porphyromonas and/or Prevotella, and especially Porphyromonas levii and
Prevotella
intermedia, and their subunits and toxins, may be used to prepare vaccine
compositions
and antibodies useful to prevent and treat bovine footrot. These organisms may
also be
used as the basis for an experimental model useful for evaluating treatments
or
preventatives for the disease. The invention is also drawn in part to the
discovery that
administration of a composition which enhances PMN phagocytosis of the
bacteria
associated with footrot is a useful method to treat or prevent footrot. Agents
which
neutralize immunoglobulin proteases and/or the causative organisms of footrot
are
especially useful.
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A. Definitions
As used herein, the following terms have the following meanings:
Adjuvant: a vehicle used to enhance antigenicity. The use of adjuvants is well-
known in the art. Adjuvant may include suspensions of minerals on which
antigen may
be absorbed, such as alum, aluminum hydroxide or phosphate; water-in-oil
emulsions
in which antigen solution is emulsified in mineral oil, such as Freund's
incomplete
adjuvant; and may include additional factors, such as killed mycobacteria in
Freund's
complete adjuvant, to further enhance antigenicity.
Antibody: a molecule, especially a protein, that binds immunologically to a
known antigen or a determinant of an antigen. Antibodies are also referred to
as
immunoglobulins, and are generally classified into isotypes (i.e., IgA, IgD,
IgG, IgE
and IgM) based on their physicochemical characteristics and amino acid
sequence
identity. IgA and IgG are further divided into subtypes (i.e., IgA,, IgAz,
IgG,, IgG2,
IgG~ and IgG4) based on sequence similarity.
Bacterin: the term used to describe vaccines containing killed or inactivated
bacteria.
Effective Amount: dose required to protect an animal against infections or
disease or alleviate a particular symptom of an infection or disease.
Footrot: also known as interdigital phlegmon, interdigital necrobacillosis,
foot
abscess, foul-in-the-foot or superfoul. A necrotizing infection of
interdigital skin
characterized by deep necrosis and fissures. It is associated with infection
with either
or both of Porphyromonas and Prevotella. The symptoms of the infection, such
as
lameness, swelling, edema, interdigital lesions, interdigital purulent
exudate, etc. are
also included in the term footrot.
Immune Response: development in the host of a cellular and/or antibody-
mediated immune response to a composition or vaccine of interest. Such a
response
may consist of one or more of the following: inducing cytokine production,
producing
antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T
cells directed
specifically to an antigen or antigens included in the composition or vaccine
of interest.
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s
Inactivation: no longer capable of causing disease or a disease condition.
Bacteria or bacterial toxins may be inactivated by many known methods. These
include, but are not limited to: chemical treatment, e.g., formalin; heat
treatment, e.g.,
mild heating until a toxin is inactivated or cells are killed; attenuation,
e.g., making a
bacterium or toxin inactive through mutation or genetic/chemical alteration;
sonication,
e.g., disruption of cells by exposing a suspension of the cells to high
frequency sound
waves; freezing, e.g., snap freezing cells until they lyse; etc.
Lactamase: also known as (3-iactamase. An enzyme or toxin which cleaves a ~i-
lactam structure such as that found in certain antibiotics. This term includes
the
lactamase produced by Prevotella intermedia.
Neutralize: able to prevent or alleviate toxic effects. An agent is a
neutralizing
agent if it prevents or alleviates the toxic effects of an organism, toxin or
subunit.
Included in this term are antibodies which bind to an organism, toxin or
subunit so as to
prevent or inhibit its toxic activity.
Porphyromonas: a genus of anaerobic bacterium that causes bovine footrot. As
used in this application, the term includes all species of this genus. The
genus
Bacteroides, formerly used to refer to as Porphyromonas, is also included in
this term
as used in this application. In particular, Porphyromonas levii was formerly
classified
as B. melanogenicus ssp.levii.
Prevention of Symptoms: includes prevention of any effect caused by an
infection or disease condition, including effects caused by a toxin.
Prevotella: a genus of anaerobic bacterium that causes bovine footrot. As used
in this application, the term includes all species of this genus. The genus
Bacteroides,
formerly used to refer to Prevotella, is also included in this term as used in
this
application. In particular, Prevotella intermedia was formerly classified as
B.
melanogenicus ssp. intermedia.
Production in vitro: production in culture, not in an infected host animal.
Production in vitro includes recombinant production.
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Protease: an enzyme or toxin which cleaves protein. Proteases may specifically
cleave certain proteins, like the IgG, specific protease produced by
Porphyromonas
levii.
Protectively Immunogenic: able to protect an animal against infection or
disease
S or alleviate particular symptoms of an infection or disease.
Recombinantly Produced: produced by means of gene expression in any other
system including microorganisms, plants or animals and/or chemically
synthesized by
methods known in the art when the gene sequence or amino acid sequence is
known.
Resolution of Infection: arrest of infection so as to return to a normal
state.
Resolution of infection includes resolution of infection mediated by
neutrophils, e.g.,
phagocytosis and oxidative metabolism.
Subunit: any part of a bacterium which is less than the whole organism.
Subunits that are antigenic may be used in vaccine compositions to produce an
immune
response. Subunits may include flagella, pili, fimbriae, membranes, membrane
proteins, toxins and any other part of an organism which may be antigenic and
induce
an immune response. This includes recombinantly produced subunits.
Toxin: a noxious or poisonous substance that is produced by a bacterium. It
may be produced and released extracellularly (exotoxin). This includes
recombinantly
produced toxins.
B. Detailed Description of the Invention
Our studies have shown that during a typical natural clinical infection,
humoral
and cellular immunity are not strongly developed toward the strains of
bacteria involved
in natural infections of bovine footrot. Minimal response is seen by Western
immunoblot to antigens from these bacteria (i.e., antibodies are poorly
produced or
readily destroyed). This may be because natural infection does not adequately
stimulate
the immune system. Alternatively, a normal immune response may be prevented by
the
infectious agent (e.g., the IgG2 specific protease produced by Porphyromonas
levii). .
Neutrophil-mediated resolution of infection includes phagocytosis of infecting
bacteria
and intracellular destruction of bacteria by oxidative metabolism.
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Bacteria have a wide variety of mechanisms for evading or altering an immune
response by a host animal. These mechanisms can enable bacteria to more
readily
establish and maintain an infection. Specifically, it is known that bacterial
proteases,
and in particular those which cleave immunoglobulins, are one such mechanism.
Such
proteases may modify important host defenses against bacterial infection.
Organisms
which produce immunoglobulin proteases include Proteus mirabilis [20,21],
Staphylococcus aureus [22], Neisseria gonorrhoeae [23] and Pasteurella
haemolytica
[24]. It is likely that bovine IgG and IgM are the most important classes of
immunoglobulins produced by cattle in response to a footrot infection. IgM is
the first
immunoglobulin recruited against a foreign antigen and IgG has the highest
serum
concentration. Both are central in immunity during tissue infections [25].
Thus,
immunoglobulin protease production by Porphyromonas levii is likely an
integral
mechanism for these bacteria to evade humoral immunity during infection.
Polymorphonuclear granulocytic neutrophils are a central component of acute
inflammation in animals [26], including cattle. These cells rapidly, and
efficiently,
phagocytose most bacteria under normal circumstances. However, we have shown
that
PMN do not readily phagocytose P. levii unless substantial amounts of specific
anti-P.
levii immunoglobulin are present. Thus, in vivo, specific anti-P. levii
immunoglobulin
is crucial for the resolution of infection.
Several types of immunoglobulin can be involved in enhancing phagocytosis by
PMN. IgG is composed of two heavy chains, each with a molecular weight of 50
kD,
and two light chains, each with a molecular weight of 25 kD. IgM, which has
two 65
kD heavy chains and two 25 kD light chains and exists as a pentamer, is a much
larger
molecule [25]. These immunoglobulin chains form distinct bands at their
respective
molecular weights upon SDS PAGE treatment. We have found the presence of bands
at
other locations after incubation with culture supernatant containing P. levii
protease,
showing that these protein chains have become fragmented. The results of our
studies
show that P. levii produces an enzyme which specifically cleaves IgG,, but not
IgG, or
IgM .
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The description of a P. levii protease which is IgG specific is significant
because
such a protease may aid in the establishment of these bacteria during an
infection by
cleaving specific IgG which could potentially aid in clearance of the
infection. In the
bovine animal IgG2 is the most important antibody in neutrophil-mediated
phagocytosis
[6] and in antibody dependent cell mediated cytotoxicity [27) . Antigen coated
with
IgG, are not readily adhered to by neutrophils or phagocytosed by these
leukocytes.
IgG2 has also been shown to mediate phagocytosis by peripheral blood monocytes
to a
greater extent than IgG,, and IgM failed to do so without complement addition.
Thus,
the action of neutrophils is an important factor in the clearance of P. levii
infection in
the bovid. By producing a protease which specifically cleaves IgGZ and
inhibiting
neutrophil-mediated phagocytosis, the organism increases its opportunities to
create a
viable infection in the bovine foot because the inability to successfully
phagocytose
infecting bacteria can compromise the host and aid in the progression of this
infection.
Thus, administration of an agent which neutralizes this protease will enhance
neutrophil-mediated phagocytosis and speed resolution of the infection by the
host.
The P. levii protease appears to function optimally at 35 °C, a
temperature not
unlike that of the distal bovine limb. The specificity of this protease
activity suggests
that the enzyme may act specifically at the hinge region of the
immunoglobulin. Such a
region is not present in IgM and is the area with the highest degree of
variance between
IgG, and IgG2 [28,29]. The near complete disappearance of heavy chain from the
IgG
preparation under conditions of lengthy incubation suggests that multiple
enzyme
activities are occurring and that the degradation of these Igs is complex.
In natural infections, neutrophils (cells that kill bacteria), but very few
lymphocytes, are recruited to the area of infection. Under optimal
circumstances,
neutrophils can resolve the infection through phagocytosis (ingestion and
destruction of
the offending bacteria). It is well known that a Th, (T-helper lymphocyte 1)
response
and the associated IgG2 that is produced by a Th, response, is critical for
neutrophils to
destroy bacteria by phagocytosis [6]. We have demonstrated that bacteria
causing
footrot (Porphyromonas levii) produce a specific toxin (a protease) that
destroys only
IgGz (see Figs. lA through 1D). This toxin may act in the foot of affected
cattle to
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destroy the IgG2 crucial for successful resolution of the infection by
neutrophils. The
use of this toxin in a vaccine composition will result in formation of
antibodies which
can neutralize or inhibit the toxin before it destroys Th, associated IgG2,
thus allowing
the neutrophils to phagocytize the bacteria.
Others [7] have postulated that immune responses can be altered and tailored
to
specific conditions (such as a Th, or a The response) through the strategic
delivery of
cytokines (immune messenger molecules). Such a tailored delivery of a vaccine
may be
useful in bovine footrot, where it would be useful to increase IgGz production
to
overcome the effects of the Porphyromonas levii protease toxin.
Bovine PMN efficiently phagocytosed P. intermedia and F. necrophorum, but
not P. levii. High titre anti-P. levii serum increased the efficiency of
phagocytosis of
P. levii and this increase in phagocytosis was independent of heat labile
complement
components. Porphyromonas levii produced an Ig protease which cleaves bovine
IgG,
but not IgM. We postulate this specific humoral immunity may be involved in
the
resolution of this infection. Porphyromonas levii produces a protease that was
found to
cleave IgG2, the Ig class involved in PMN-mediated phagocytosis, but not IgG,
or IgM.
This enzyme is, thus, a significant virulence factor in AIP through local
inhibition of
immunoglobulin assisted PMN-mediated phagocytosis.
We have discovered in our PMN phagocytosis studies that the action of
neutrophils is an important factor in the clearance of P. levii infection in
the bovid
affected with acute interdigital phlegrnon. By producing a protease, or
possibly several
proteases, which act alone or collectively to specifically cleave IgG2 and
therefore
inhibiting PMN-mediated phagocytosis, P. levii increases its opportunities to
create a
viable infection in the bovine foot. The inability to successfully phagocytose
these
bacteria could compromise the host and aid in the progression of this
infection. Thus,
agents which neutralize such protease are useful to treat footrot.
Studies from our laboratory show that PMN chemotactically move toward
bacteria associated with acute footrot in an efficient manner, demonstrating
that their
motility is unaffected by these bacteria. Results are presented in Table A.
Chemotaxis
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is the movement of cells (here, PMN's) along a concentration gradient of the
agent,
while chemokinesis is the movement of PMN along a gradient of serum.
Table A. Neutrophil Chemotaxis and Chemokinesis
Stimulus Chemotaxis (wm/h)Chemokinesis (~cm/h)
Negative Control 11.2 + 0.5 94.1 + 5.6
Positive Control 24.8 + 0.9 121.4 + 1.5
P. levii 22.8 + 2.1 122.6 + 1.8
P. intermedia 29.7 + 2.0 80.7 + 2.3
F. necrophorum 44.8 + 1.0 99.3 + 2.3
All 3 Bacteria 32.1 + 2.3 110.7 + 2.8
Additionally, we have found that PMN demonstrate significant oxidative
metabolism when exposed to P. levii or F. necrophorum, but that the percentage
of
IS PMN which are oxidatively metabolic decreased when exposed to P.
intermedia. Thus,
decreasing oxidative metabolism of PMN by Prevotella intermedia is likely an
integral
mechanism for these bacteria to evade host resolution of infection. Results
are
presented in Table B.
Table B. PMN Oxidative Metabolism
Experimental Group % PMN Oxidatively Active
(MeanSE) n=9
Resting 10.00.6
Stimulated {PMN) 94.0 t 0.5
F. necrophorum 80. 8 1.2
P. levii 82.8 t0.7
P. intermedia 18.30.7
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Prevotella intermedia also produces a toxin (a ~3-lactamase enzyme) that
destroys certain antibiotics, i.e., those which contain a (3-lactam ring
structure, such as
penicillins and cephalosporins (Fig. 2). This enzyme has been partially
purified by
precipitating the enzyme in 40-50% ammonium sulfate (Fig. 3). Use of this
enzyme in
a vaccine composition will result in raising antibodies against the enzyme
which
inactivate its activity. This will allow more efficient use of antibiotics,
even ~3-lactam
antibiotics, to treat the infection.
We have shown via immunoblots with acute and convalescent sera of
experimentally infected animals that one can successfully immunize cattle with
antigens
of Porphyromonas and Prevotella (Figs. 4A through 4C). Acute and convalescent
sera
from some feedlot steers with footrot show rising titers to these antigens
(Fig. SA
through SD), but the reaction is not as strong as that seen in experimentally
infected
steers. These Western blots indicate that antibodies are weakly or not at all
produced
against Fusobacterium necrophorum, further supporting a minimal role for this
bacterium in the disease.
Moreover, histological assessment of experimentally infected feet indicates
that
predominantly macrophages (phagocytic and antigen presenting cells) and
lymphocytes
(antibody producing and processing cells that coordinate immunity) are
present. These
data show that Porphyromonas and Prevotella vaccine compositions provide
effective
humoral and cellular immune responses against footrot-causing organisms. Pre-
exposure vaccination with vaccine compositions prepared from these organisms
and/or
their subunits and/or toxins provide antibody, immune memory cells, and other
specific
lymphocytes which will alter the course of infection through enhancing the
function of
neutrophils. The most effective treatment must negate the activities of the
Porphyromonas levii IgG2 toxin to allow neutrophil destruction of bacteria.
This invention provides vaccine compositions comprising the footrot causing
bacteria Prevotella and Porphyromonas. It has been found, using surgical
biopsy
methodology to obtain specimens from bovine footrot, that these bacteria, and
not
Fusobacterium necrophorum which was traditionally thought to cause bovine
footrot,
are the causative agents of footrot. We have used these bacteria, grown under
strictly
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anaerobic conditions in the laboratory, in the presence of haemin and Vitamin
K, to
vaccinate animals. Further, we are able to produce and isolate bacterial
toxins from in
vitro cultures of these bacteria. These toxins can be used for protective
immunization
against infection by the bacteria causing bovine footrot or for raising toxin
neutralizing
antibodies for use in passive immunization of affected animals.
In particular, Porphyromonas levii and Prevotella intermedia, which are
present
in the cellulitic tissues of animals affected with bovine footrot, are
preferred for use in
the present invention. In a most preferred embodiment, the invention provides
inactivated bacteria, toxins, and antibodies to toxins and antigens of
Porphyromonas
levii and Prevotella intermedia. Such inactivated bacteria, toxins, and
antibodies are
useful for preventing and treating bovine footrot and the symptoms thereof.
A variety of strains of Porphyromonas and Prevotella are useful in the present
inventions. Strains which grow well in vitro, are able to infect the target
animal
species, and produce toxin when grown in vitro, are preferred. The bacterial
strains
used in this invention are cultured by growing them in appropriate media,
e.g., bovine
tissue based medium, such as chopped meat medium or brain heart infusion
medium
that has been supplemented with haemin and Vitamin K, with resazurin as an
indicator,
under conditions of strict anaerobiasis.
The invention also provides compositions comprising inactivated Prevotella
and/or Porphyromonas bacteria which compositions are protectively immunogenic.
Various strains of Prevotella and Porphyromonas bacteria may be useful in such
vaccine compositions. Vaccine compositions may contain one or more of the
bacteria
causing bovine footrot, and/or one or more subunits and/or toxins of these
bacteria.
Porphyromonas levii and Prevotella intermedia are preferred. In particular,
strains of
Porphyromonas levii and Prevotella intermedia which produce large amounts of
toxin
when cultured in vitro are most preferred.
The bacterial strains may be cultured as set forth in the Examples below, then
harvested for use in vaccine compositions. Bacteria are preferably inactivated
before
use in vaccine compositions. Various methods of disruption may be preferably
used to
inactivate the bacteria, including sonication, osmosis, use of pressure
differentials or
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freezing. Freezing is the most preferred. Conventional techniques such as mild
heat
treatment or formalin inactivation may also be used to inactivate bacteria or
toxins used
in vaccine compositions.
The formulation of such vaccine compositions may include suitable
pharmaceutical carriers, including adjuvants. The use of an adjuvant, such as
an alum-
based adjuvant is preferred. Many commercial adjuvants may be useful in the
present
invention. For these studies an alum-based adjuvant containing aluminum
hydroxide
(Imject Alum, Rockford, IL) was used. Exact formulation of the vaccine
compositions
will depend on the particular formulation and the route of immunization. Such
vaccine
composition formulation is well-known to those skilled in the art [30].
Such vaccine compositions are useful for immunizing any animal susceptible to
footrot. The present invention provides a method of preventing or treating
footrot by
administering an effective amount of a vaccine composition of Prevotella
and/or
Porphyromonas to an animal in need of such prevention or treatment. Bovines
are
preferable treated. In particular, preferred vaccine compositions comprise
Porphyromonas levii and/or Prevotella intermedia.
The invention further provides compositions comprising neutralizing agents,
such as antibodies, to Prevotella and/or Porphyromonas bacteria and/or their
toxins or
subunits, which compositions are useful to treat footrot. Formulation of such
compositions may include suitable pharmaceutical carriers, and is also well-
known to
those skilled in the art.
The route of administration may be any convenient route, and may vary
depending on the particular bacteria, subunit, toxin or agent used in the
composition,
the animal to be treated and/or vaccinated, and other factors known to those
of skill in
the art. Parenteral administration, such as subcutaneous, intramuscular, or
intravenous
administration is preferred. Subcutaneous administration is most preferred for
food-
producing cattle. Oral administration may be used, including oral dosage forms
which
may be enteric-release coated. Intraperitoneal, nasal and rectal routes of
administration
are also contemplated.
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The schedule of administration may vary depending on factors such as the
bacteria and the animal being treated. Animals may receive a single dose, they
may
receive an initial dose and a booster dose, or they may receive multiple
doses. Annual
boosters may be used for continued protection. In particular two doses 21 days
apart
are preferred as a primary course. Development of such schedules of
administration is
known to those of skill in the art.
The age of the animal to be treated may also affect the route and schedule of
administration. For example, for vaccination, administration is preferred at
an age
when maternal antibodies are no longer present and the animal is
immunologically
competent. This is about 2-4 months of age in cattle. Additionally,
administration of
vaccine composition to mothers so that they develop antibodies is useful to
prevent
infection of their offspring through passive transfer of antibodies in
colostrum and
milk.
The methods of this invention are effective in preventing colonization of the
subcutaneous tissues of the foot with bacteria which cause bovine footrot.
They are
also effective in preventing the symptoms of bovine footrot. This includes
neutralization of toxin and prevention of the physiological effects of toxin
which may
occur when Porphyromonas and/or Prevotella are present in the foot of the
animals.
Treatment may be administered to symptomatic or asymptomatic animals,
including animals with chronic infection. Inducing the production of
neutralizing
antibody against Porphyromonas protease toxin or delivering antibody or
another agent
which neutralizes this toxin allows natural development of neutrophil
associated
antibody that provides successful recovery from infection with footrot causing
organisms such as Porphyromonas and/or Prevotella. Such antibodies or other
neutralizing agents will also allow development of antibodies to other
organisms (such
as Fusobacterium necrophorum) which may be associated with footrot.
The present invention also provides toxins of Porphyromonas levii and
Prevotella intermedia and methods of producing these toxins. It has been found
that
these organisms, when cultured in medium with haemin and Vitamin K under
anaerobic
conditions, produce certain toxins in vitro. These toxins may be used to
immunize
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animals against footrot. The toxins may be isolated from the supernatants of
the
bacterial cultures. They are useful for preventing and treating infection.
Supernatants from the cultured bacteria (Porphyromonas levii) contain a toxin
(protease) that specifically cleaves bovine IgGz into fragments. Isolated
toxin was
S incubated with biotin-labeled immunoglobulin and the resulting mixture
separated by
SDS PAGE. When active toxin was present, IgG, fragments were produced as the
toxin cleaved the immunoglobulin. Immunoglobulins remained intact when no
toxin
was present or when the toxin was purposely inactivated. The toxin was also
shown
not to be active against other immunoglobulins (i.e., IgG, and IgM). Figs. lA
through
1D present these results.
Supernatants from the cultured bacteria (Prevotella intermedia) contain a
toxin
(~i-lactamase enzyme) that specifically cleaves (3-lactam antibiotics. See
Fig. 2. This
toxin has been purified using ammonium sulfate precipitation methods.
The present invention also provides antibodies to Prevotella and Porphyromonas
and/or their toxins or subunits. Polyclonal and monoclonal antibodies may be
raised by
conventional techniques. These antibodies will be useful to enhance neutrophil-
mediated phagocytosis and as ~n antiserum to neutralize the effects of toxin.
Thus,
when administered to an animal, e.g., intravenously, they may be expected to
relieve
symptoms of bovine footrot.
Two strains of anaerobic bacteria, Porphyromonas levii and Prevotella
intermedia, were assessed for their ability to induce acute footrot in cattle.
They were
used in conjunction with Fusobacterium necrophorum. Cattle feet were
experimentally
infected with these bacteria and animals were examined clinically,
histopathologically,
and bacteriologically for response to inoculation with these anaerobic
bacterial species.
After experimental induction of footrot, response to a macrolide antibiotic to
evaluate
its potential usefulness as a footrot treatment was examined. Porphyromonas
levii
caused a significantly more severe infection than Prevotella intermedia, as
assessed by
clinical scoring, when inoculated into the bovine interdigital space.
Histological
examination of affected tissue demonstrated local edema, necrosis,
infiltration of the
subcutaneous tissue with leukocytes, and bacterial cells morphotypic of the
strains used
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to infect the interdigital tissues. In all cases bacteria recovered from
biopsy samples of
the infected feet six days following experimental infection were consistent
with those
strains initially injected into the animals. MIC's and MBC's of bacteria used
to infect
feet were not different from the MIC's and MBC's of bacteria recovered from
the feet
six days later, but prior to therapy. All animals were treated with antibiotic
on the
sixth day following the experimental infection and 83.3 % (5/6) responded very
favorably to this treatment, based on a subjective assessment of the severity
of
lameness, swelling, and lesions.
Cattle injected in the interdigital skin and subcutaneous tissue with
Porphyromonas levii or Prevotella intermedia, together with Fusobacterium
necrophorum, developed a clinical condition consistent with acute footrot
within five
days, causing interdigital necrosis, swelling of the interdigital tissues and
distal
subcutaneous tissue, and lameness. This experimental model resulted in lesions
and
lameness typical of the natural infection in cattle. Histopathological
features (edema,
leukocyte infiltration, necrosis) in the subcutaneous tissues were also
typical of natural
infection. All of these findings are considered characteristic of acute
footrot. We have
demonstrated that Porphyromonas levii or Prevotella intermedia inoculated into
the
interdigital skin of cattle can induce acute footrot, in particular when
injected in
conjunction with Fusobacterium necrophorum.
This experimental model infection system is useful to further examine the
pathophysiological and immunological consequences of acute bovine footrot and
is a
useful screening tool for the evaluation of potential new therapeutics for
footrot, such
as antibiotics and vaccines. Based on these results, it appears that the
macrolide
antibiotic we tested is a useful antibiotic for the treatment of acute bovine
footrot.
C. Examples of Embodiments of the Invention
The following examples are not intended to limit the scope of the invention in
any manner. In general the following materials and methods were used in the
examples
unless otherwise noted:
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1. Medium Preparation.
The medium used for culture of the Porphyromonas levii and Prevotella
intermedia (Brain Heart Infusion broth with haemin and Vitamin K and
resazurin) was
prepared as indicated below.
Medium Com op vent
Calf brains, infusion from 200 g
Beef heart, infusion from 250 g
Bacto protease peptone 10 g
Bacto dextrose 2 g
Sodium chloride 5 g
Disodium phosphate 2.5 g
Hemin 5 mg
Vitamin K 1 mg
Resazurin 1 mg
2. Biopsy Isolation of Porphyromonas levii and Prevotella intermedia.
The foot of a bovine with clinical footrot was lifted and cleaned using water
and
disinfectant solution followed by 70:30 (vol/vol) ethanol:water. A small
incision was
made using a scalpel blade (#10) over a swollen portion of the limb
immediately
proximal to the affected region of the foot. A TrucutT"" hepatic biopsy needle
was
inserted into the subcutaneous tissue. A core of tissue was collected, placed
in pre-
reduced transport medium (Anaerobe Systems, San 3ose, CA} and transported
immediately to the laboratory in anaerobic transport medium.
Porphyrombnas levii and Prevotella intermedia were grown separately on
kanamycin:vancomycin laked blood agar and Brucella blood agar for five days
under
conditions of 5 % HZ:S %C02:90% NZ at 37°C. A single colony of each
organism was
inoculated into a separate tube of modified chopped meat medium and grown for
24h at
37°C in an anaerobic chamber.
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3. Freezing Porphyromonas levii and Prevotella intermedia.
The isolated bacteria were kept frozen at -85 °C in either pre-reduced
transport
medium or semisolid BHI broth under anaerobic conditions.
4. Growing Porphyromonas levii and Prevotella intermedia.
Both bacterial species were grown in supplemental BHI broth (above). Broths
were inoculated from secondary cultures that have been expanded from primary
cultures stored in the laboratory. Bacteria were incubated at 37°C in
an anaerobic
atmosphere (5 % carbon dioxide, S % hydrogen, balance nitrogen). Broths of
Porphyromonas levii were incubated 96h and Prevotella intermedia broths were
incubated 72h.
5. Harvesting Porphyromonas levii and Prevotella intermedia.
Bacteria were harvested by centrifugation at 3000 x g for 10 min. Pellets were
resuspended and washed three times in sterile PBS.
fi. Preparing Vaccine from Porphyromonas levii and Prevotella intermedia.
To lyse cells the washed bacteria were snap frozen in liquid nitrogen, thawed,
and snap frozen again. Protein concentration was determined by the Bradford
protein
assay and vaccines were stored at -20°C until needed.
EXAMPLE 1
Immunological Assessment of Animals Naturally Infected
with Bovine Footrot
To assess the humoral immunity (antibody production) resulting from natural
infection with bovine footrot, acute and convalescent sera were obtained from
affected
animals. Acute serum was collected via jugular venipuncture on the day an
animal
initially showed clinical signs of acute bovine footrot (i.e., lameness,
swelling of the
interdigital region and distal foot, and/or a necrotic lesion of the
interdigital space).
Twenty-eight days following appearance of clinical signs, a second
(convalescent)
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sample was collected, also by jugular venipuncture. Serum was separated from
cells
and stored at -8S °C until used in the Western blotting assays.
Porphyromonas levii (7-S) was grown on Brucella blood agar and inoculated
into pre-reduced chopped meat medium with Vitamin K, hemin and resazurin and
S grown anaerobically (S % HZ:S %C0z:90% NZ) for 24h. Prevotella intermedia (S-
2) was
grown under similar conditions for 48h and Fusobacterium necrophorum ATCC
27852
(ovine footrot isolate) was grown for five days on chocolate agar under
similar
conditions, inoculated into modified chopped meat medium and grown
anaerobically for
24h. Bacteria were harvested by centrifugation (5000 x g for 1S min) and the
pellets
placed in frozen aliquots. Protein concentrations of these aliquots of
centrifuged
bacterial cells following a freeze-thaw were determined by the Bradford
protein assay.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was
conducted on these bacterial specimens (10 ~,g protein per lane) following
boiling in
SDS PAGE reducing and denaturing sample buffer according to accepted
methodology
1S [31 ] . Electrophoretically separated proteins within the gel (4 % stacking
gel and 12
resolving gel) were electrophoretically transferred to nitrocellulose paper
(18h at 4°C
and 30V) according to accepted methodology [32]. Protein transfer was
confirmed by
amido black staining of a strip of nitrocellulose paper. Strips were blocked
with 1
skim milk in Tris buffered saline and exposed to either acute serum or
convalescent
serum from cattle with footrot. Sera were used at the same dilution (1:200). A
monoclonal antibody raised against bovine IgG (heavy chain specific)
conjugated to
alkaline phosphatase was used as a detection method for bound bovine IgG.
Color
reaction involved incubation of nitrocellulose strips with nitro blue
tetrazolium in 0.2M
Tris-HCl 4mM MgCl2 pH 8.8 buffer with S-bromo-4-chloro-3-indolyl phosphate in
2S dimethyl sulfoxide. Development of blots proceeded for exactly 10 min and
the
development reaction was stopped with ice water. Blots were immediately
photographed.
Western blotting results from four animals are shown in Figs. SA through SD.
In each figure, the acute (A) and convalescent (C) serum reactions are shown
against
the antigens from Fusobacterium necrophorum ATCC 27852, Prevotella intermedia
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(5.2) and Porphyromonas levii (7.5). In animal 420308 (Fig. SA), increased
titers of
IgG antibodies against Prevotella intermedia (5.2) and Porphyromonas levii
(7.5) were
seen in convalescent serum relative to acute serum. Only a slight increase in
antibody
titer was seen with Fusobacterium necrophorum ATCC 27852. In animals 1875RED
(Fig. SB), 2281GRN (Fig. SC), and PURO1 (Fig. SD), no rising titers of IgG
against
Fusobacterium necrophorum were detected, but increasing IgG titers to one or
both of
Prevotella intermedia or Porphyromonas levii were clearly observed. This is
evidenced
by the increasing number and intensity of the protein bands observed on the
strips in
the convalescent sera samples. These data show that over the 28-day period in
which
the animals recovered from the disease, a specific immune response was
produced
against Prevotella intermedia (5.2) and/or Porphyromonas levii (7.5), but no
substantive production of IgG specifically reactive to Fusobacterium
necrophorum was
detected. These data indicate Fusobacterium necrophorum is not immunologically
recognized by the host. They also indicate that Prevotella intermedia and/or
Porphyromonas levii are important agents in the disease, and that a
comparatively weak
(1:200 dilutions) serum IgG response is produced toward these bacteria in the
course of
natural infection.
EXAMPLE 2
Immunologicat Assessment of Animals Experimentally Infected
with Bovine Footrot
To assess the humoral immunity (antibody production) resulting from
experimental infection with bovine footrot, pre-exposure serum, acute serum,
and
convalescent sera were obtained from animals experimentally infected with
bovine
footrot as described in Example 5 below. Pre-exposure serum was collected
prior to
infection by jugular venipuncture. Acute serum was collected by jugular
venipuncture
on the day following experimental infection and a second (convalescent) sample
was
collected, also by jugular venipuncture, 11 days following infection. Serum
was
separated from cells and stored at -85 °C until used in the Western
blotting assays.
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Porphyromonas levii (7-5), Prevotella intermedia (S-2) and Fusobacterium
necrophorum ATCC 27852 (ovine footrot isolate) proteins were prepared as
described
in Example 1.
Western blotting results (performed as described in Example 1) from a steer
S experimentally infected with Porphyromonas levii and Fusobacterium
necrophorum are
shown in Figs. 4A through 4C. In Fig. 4A the antigen used was Fusobacterium
necrophorum ATCC 27852. The results shown in Fig. 4A clearly demonstrate that
levels of IgG against F. necrophorum are negligible at all time periods tested-
pre-
exposure, acute exposure and convalescent exposure. Conversely, this animal
showed
rising IgG titers to both Prevotella intermedia 5.2 (Fig. 4B) and
Porphyromonas levii
7.5 (Fig. 4C), indicating some shared antigenicity between Prevotella
intermedia 5.2
and Porphyromonas levii 7.5. The negligible immune response to Fusobacterium
necrophorum suggests that it is not readily recognized by the host immune
system
and/or that it is not involved in the active infection of the subcutaneous
tissues in
footrot.
Four of the six experimentally infected animals were re-assessed as above
approximately eight weeks following experimental infection to determine the
presence
of serum IgG specific for whole bacterial cell extract antigens. The pattern
of serum
IgG remained the same. This demonstrates that the serum IgG produced in
response to
experimental infection lasts for a minimum of several weeks and similar
lasting serum
IgG may be created via vaccination. The data show that, despite the known
presence of
Fusobacterium necrophorum within experimentally infected tissue, no
substantial
immunological response to this bacterium occurs in experimentally infected
cattle,
while Prevotella intermedia and/or Porphyromonas levii are the important
etiologies of
the disease.
EXAMPLE 3
Production and Assessment of Immunoglobulin G2 Destroying Toxin
N-hydroxy-succinomidobiotin (Sigma Biosciences, St. Louis, MO) was
dissolved in dimethyl sulfoxide to a 1 mg/ml concentration. It was then added
to equal
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one tenth the weight of bovine IgG (Sigma Biosciences, St. Louis, MO), IgM
(Sigma
Biosciences, St. Louis, MO), IgG, (Chemicon International, Inc., Temecula. CA)
and
IgG, (Chemicon International, Inc., Temecula, CA). The solution was then
incubated
for 2h at 25°C and the reaction stopped with 1 mg of glycine (Sigma
Biosciences, St.
S Louis, MO). The biotinylated immunoglobulins were dialyzed overnight in
three
changes of double distilled water. Final concentrations were 10 mg/ml for IgG,
IgG,
and IgGz and 1.1 mg/ml for IgM.
Supernatant of a broth culture of P. levii grown in anaerobic conditions (5
H2:5% C0~:90% Nz) in Cooked Meat broth (BBL, Cockysville, MD) (supplemented
with 5 mg/1 of hemin, 1 mg/1 of Vitamin K and 1 mg/I resazurin (Sigma
Biosciences,
St. Louis, MO) at 37°C for four days was collected by centrifugation.
The bacterial
culture was centrifuged at 3020 x g for 20 min to remove bacterial cells. The
remaining supernatant was placed in aliquots of 250 ~,l amounts for the
protease assay.
Culture purity was assured by streak plating onto Brucella Blood Agar and
standard
bacteriological identification.
Procedures were slight modifications of the methods of Lee and Shewen [24].
Ten microliters of biotinylated immunoglobulin was added to 250 ~,1 of
bacterial culture
supernatant and 2.5 p.l of penicillin-streptomycin solution. The mixture was
incubated
at 35°C and 10 ~,1 samples taken at 0, 0.5, 1, 2, 4, 8, 24, 48, and
72h. The samples
were added to a 10 ~l of sample buffer, boiled for 4 min and analyzed by SDS
PAGE,
electrophoretic transfer and streptavidin-alkaline phosphatase exposure.
Controls
included P. levii supernatant heated to 100°C prior to exposure to the
biotinylated Igs
and uninoculated culture supernatant.
All samples were loaded onto 15 % resolving gels with 4 % stacking gels for
SDS PAGE using standard methodology [31). Gels were run on ice at 150V for 15
min
and at 200V for 45 min in 10% running buffer. The proteins in the gel were
then
transferred to nitrocellulose paper using standard methods [32). Transfer was
at 20V
overnight at 4°C. Blocking was conducted using a lh incubation at
37°C in a solution
of skim milk powder (2 % wt/vol) and Tris buffered saline containing Tween 20
( 18g
NaCI, 2.42g Trizma base, 2 1 double distilled water, 1 ml Tween 20, pH 7.4)
(TBS-
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tween). Blots were washed with TBS-tween three times (15 min) at room
temperature,
then incubated in a 1:5000 dilution of streptavidin-alkaline phosphatase
(Sigma
Biosciences, St. Louis, MO) in TBS-tween for 2h at 37°C. The blots
were again
washed three times for 15 min in TBS-tween. Nitroblue tetrazolium (Sigma
Biosciences, St. Louis, MO) (NBT) and 5-bromo-4 chloro- 3 indolyl phosphate
(Sigma
Biosciences, St. Louis, MO) (BCIP} were added as substrate for color
development,
which was allowed to proceed for 4 min. Substrate solution was composed of 10
mg of
NBT dissolved in 100 ml of developing buffer (31.52g Tris-HCL, 0.18g MgCI,-
6H20>
1 1 distilled water, pH 8.8) and 5 mg of BCIP dissolved in 1 ml of dimethyl
sulfoxide
was added to this solution. Protein bands representing the light and heavy
chains of the
biotinylated immunoglobulins, or fragments of these immunoglobulin chains were
viewed based on alkaline phosphatase activity on the nitrocellulose paper.
Photographs
were obtained immediately.
Assays performed on biotinylated IgG produced degradation bands, fragments
of the immunoglobulin heavy and light chains, by O.Sh, as seen in Fig. lA. The
intensity of these Ig fragment bands continued to increase during the 72h of
incubation.
Optimization studies performed on IgG showed that incubation of the
supernatant with
IgG at 35 °C showed the most pronounced activity and that a
streptavidin-alkaline
phosphate dilution of 1:5000 gave optimal results. All further assays were
performed
under these same conditions. When IgM was examined using these methods (Fig.
1B)
there were no degradation bands (immunoglobulin fragments) formed. Only bands
representing intact heavy and light chains of IgM could be visualized. When
IgG, was
assayed, degradation bands were not evident (Fig. 1C) and again only bands
representing intact heavy and light immunoglobulin chains could be recognized.
However, when assays using IgG2 were conducted results similar to those
performed
with mixed subtype IgG were seen. Degradation bands were evident within 30
min, as
demonstrated in Fig. 1D. Controls using heated supernatant and uninoculated
media,
each of which was exposed to the biotinylated bovine IgG, showed no evidence
of
degradation bands. This indicated that fragmentation of the immunoglobulins
was the
result of Porphyromonas levii enzyme activity.
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EXAMPLE 4
Production and Assessment of ~i-Lactamase Enzyme Toxin
The major mechanism of resistance to penicillin and other (3-lactam
antibiotics is
the presence of a group of bacterial enzymes termed ~3-lactamases. Since their
first
discovery in the 1940's [33], the widespread use of penicillins is thought to
have led to
an increased occurrence of ~i-lactamases [34]. (3-lactamases are enzymes
capable of
hydrolyzing ~3-lactam antibiotics such as the penicillins and the
cephalosporins. They
can be chromosomally mediated or plasmid mediated and constitutive or
inducible [35] .
(3-lactamase inhibitors, such as clavulanic acid or sulbactam, can be used to
prevent the
~i-lactamase from inactivating the ~i-lactam antibiotic, but these inhibitors
have slightly
different inhibition profiles [36], so the choice of which inhibitor to use
will depend on
the specific type of ~3-lactamase present. Specific immunity raised against
the enzyme
may allow more successful therapy.
Antibiotic Susceptibility Tests: Four anaerobic bacterial isolates of
Prevotella
intermedia were retrieved from storage in Amies medium at -85 °C and
plated onto
Brucella blood agar (BBA) plates. Penicillin resistance was tested using a
disc
diffusion method. Colonies from BBA plates were suspended in vials of sterile
saline
(0.85 % NaCI} to reach a turbidity roughly equal to tube 3 of the McFarland
turbidity
standards (approximate equivalent density of 9.0 x 10~ bacteria/mL). One (1)
mL of
that suspension was transferred to a fresh BBA plate and spread equally around
the
plate to create a lawn of bacteria. The plate was allowed to dry before
applying two
antibiotic discs to the surface of the agar; one disc contained 2 units of
penicillin G (P2)
and the other contained 10 units (P10). The plate was incubated overnight in
an
anaerobic chamber at 37°C and the diameters of any zones of inhibition
were
measured.
~i-Lactamase Testing: Any of the above bacterial isolates showing resistance
to
penicillin G were tested for (3-lactamase activity using commercially
available nitrocefin
impregnated filter paper discs (Cefinase - BBL). Some results using assay are
demonstrated in Fig. 2. Colonies from the antibiotic susceptibility plates and
from
normal BBA plates were tested to determine if the (3-lactamase activity was
induced by
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antibiotic exposure. Colonies removed from an agar plate were smeared onto the
disc
surface and the disc was observed for a color change for up to Ih. If the disc
changed
from yellow to red in color, it was considered to be a positive result, while
no color
change indicated a negative result. Note that for testing broths or other
liquids, a drop
of the fluid to be tested was added to the dry disc and then observed for a
color change.
Enzyme Isolation: The Prevotella intermedia isolates exhibiting ~i-lactamase
activity were used to isolate the (3-lactamase itself. As previously outlined,
the bacteria
of interest were grown in Brain Heart Infusion broth cultures supplemented
with
0.005 % hemin and 0.5 % yeast extract. Three hundred fifty (350) mL broth
cultures
were made anaerobic by bubbling nitrogen gas through them before autoclaving
with
tightly sealed caps. The cultures were inoculated, then left to incubate at
37°C in an
anaerobic chamber for 48h. Following incubation, the broth contents were
tested for (3-
lactamase activity, and a BBA plate was made to check the purity of the
culture. The
cultures were centrifuged at 5,000 x g for 15 min at 4°C. The
supernatant was
discarded and the pellets resuspended in 50 mM phosphate buffer, pH 7 and
centrifuged
at 15,000 x g for 40 min at 4°C. This washing procedure was repeated
once more and
the resulting pellet (resuspended in 50 mM phosphate buffer) was sonicated at
60
maximum with 6-7 bursts of 30 seconds each, to disrupt the cells. The sonicate
was
centrifuged at 27,000 x g for lh at 4°C and the supernatant retained
for ammonium
sulfate precipitation. ~3-lactamase activity was periodically re-checked
throughout the
enzyme preparation.
Ammonium Sulfate Precipitation: The supernatant from the above procedure
was subjected to ammonium sulfate precipitation [37] to begin the purification
process.
Sufficient ammonium sulfate was initially added to achieve 40% saturation and
the
mixture was stirred on ice for 20 min. This was then centrifuged at 10,000 x g
for 20
min at 4°C. The pellet was resuspended in 50 mM phosphate buffer and
saved as the
40% saturation fraction, while the supernatant was used further in the
precipitation.
Taking into account how much ammonium sulfate had already been added. enough
ammonium sulfate was added to bring the supernatant to 50 % saturation. This
was
stirred over ice for 20 min then centrifuged at 10,000 x g for 20 min at
4°C. The pellet
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was resuspended and saved as the 40-50 % saturation fraction. The procedure
was
repeated once more for 60 % saturation, after which the resuspended pellet was
saved at
the 50-60 % fraction and the supernatant was saved as the greater than 60 %
fraction. . .
The ammonium sulfate was removed from the four fractions by overnight dialysis
using
dialysis tubing with a molecular weight cut-off of 25,000 Daltons in a flask
with
distilled water constantly flowing through. The samples were then tested for
(3-
lactamase activity and those fractions testing positive were used as enzyme
isolates.
Gel Electrophoresis: The isolated enzyme fraction was run on a 7 % SDS
PAGE, and then stained with silver stain to visualize proteins in the sample.
Antibiotic susceptibility tests were performed on the bacterial isolates.
Results
are shown in Table 1. Prevotella intermedia isolates 5-2, 8-2 and 14-7 showed
zones
of what has been called regrowth. There was indeed a zone of inhibition, but
within
that zone of inhibition, there was re-growth immediately around the antibiotic
disc.
Bacteroides fragilis (7-6) was clearly resistant to penicillin.
Table 1. Zones of Inhibition of Isolates from
Bovine Footrot Tissue Samples
Isolate Identification P2 zone of inhibition (mm) P10 zone of inhibition (mm)
Prevotella intermedia22 (with regrowth) 28 (with regrowth)
(5-2)
Bacteroides fragilis 0 p
Prevotella intermedia27 (with regrowth) 35 (with regrowth)
(8-2)
Prevotella intermedia34 (with 22mm regrowth)38 (with 23mm regrowth)
(14-7)
(3-Lactamase Testing results are shown in Table 2, and demonstrate that
Prevotella intermedia isolates 5-2, 8-2 and 14-7 produced (3-lactamase.
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Table 2. ~3-Iactamase Test Results from Footrot Isolates
Grown on Two Media Types
Isolate Identification Antibiotic Susceptibility Plate BBA Plate
Prevotella intermedia positive positive
(5-2)
Bacteroides fragilis strongly positivestrongly positive
Prevotella intermedia positive positive
(g-2)
Prevotella intermedia positive positive
( 14-7)
Isolates of Porphyromonas levii were found to be sensitive to the antibiotic
penicillin G and were (3-lactamase negative.
Enzyme activity of the ammonium sulfate precipitation fractions from strain 7-
6
(B. fragilis) was found in the 50-60 % fraction, but with 5-2 (P. intermedia)
the enzyme
was found in the 40-SO % fraction.
Results of gel electrophoresis are illustrated in Fig. 3. These fractions
contained enzyme activity, although the activity has not yet been attributed
to any single
protein band within the fractions.
EXAMPLE 5
Experimental Infection Model
Experimental Animals: Yearling Canadian Simmental crossbred steers (400 kg)
were used for this investigation (n=6). Each animal was individually
identified with an
eartag and commingled in a large pen. Steers were fed a diet of good quality
hay ad
libitum and fresh water. Animals were obtained from a single source and held
for
seven days prior to the initiation of the study. Animals were examined by a
veterinarian to ensure suitability for the study. All procedures were
conducted
according to the guidelines of the Canadian Council on Animal Care.
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Growth of Bacteria: Porphyromonas levii and Prevotella intermedia, both of
which were isolated from the subcutaneous tissues of feedlot animals affected
with
acute footrot, were grown separately on kanamycin:vancomycin faked blood agar
for
five days under conditions of 5 % H2:5 % C02:90 % NZ at 37 °C. A single
colony of each
organism was inoculated into modified chopped meat medium (BBL) and grown for
24h
at 37°C in an anaerobic chamber. Fusobacterium necrophorum (ATCC 27852)
was
grown anaerobically for five days on chocolate agar, and a single colony was
inoculated
into modified chopped meat medium and grown under anaerobic conditions for
24h, as
described above. Samples of washed cells from broth culture were serially
diluted and
spread on agar to determine the precise number of viable bacteria (CFU/ml) in
each
suspension of bacteria. All manipulations were conducted under anaerobic
conditions
and these bacteria were not exposed to air until immediately prior to
injection into the
interdigital region of the steers.
Experimental Infection: The animals were individually restrained in a head
gate
and squeeze apparatus and the left hind foot lifted and restrained with rope.
The
interdigital region of the foot was carefully cleaned with water and a
disinfectant
solution and rinsed with 70% ethanol:water (vol:vol). Local anaesthesia was
employed
using lidocaine and the interdigital skin was excoriated using mechanical
abrasion.
Porphyromonas levii and Fusobacterium necrophorum, or Prevotella intermedia
and
Fusobacterium necrophorum, were drawn into a common syringe. This mixture (1.0
ml), containing 109 of each of the two organisms, was injected into the
interdigital skin
and subcutaneous tissue (0.5 ml per location). Three steers received
Porphyromonas
levii mixed with Fusobacterium necrophorum (Group 1) and three steers received
Prevotella intermedia mixed with Fusobacterium necrophorum (Group 2). The
animals
were monitored closely after experimental inoculation for any immediate
adverse
reactions and daily thereafter.
Clinical Scoring: The severity of clinical signs present in each animal
following
experimental infection was monitored and scored using a slight modification of
a
previously published scoring system [17]. Briefly, this involved daily
examination of
the animal and subjective assessment of the lameness, lesion, and swelling.
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Lameness was evaluated daily as:
0 = normal
1 = slight lameness - puts some weight on foot but moves readily
2 = moderate lameness - does not want to put weight on foot and moves
slowly
3 = severe lameness - holds foot up at intervals and is reluctant to move or
place weight on the foot (prefers to lie down)
Swelling was evaluated and scored daily as:
0 = no swelling
1 = slight to moderate swelling
2 = moderate to severe swelling
3 = severe swelling
Lesions were evaluated and scored daily as:
0 = no lesions
1 = lesion healed or healing
2 = small interdigital lesion extending up to 1/ to '/2 length of the
interdigital space
3 = very large necrotic lesion involving almost all of the interdigital space
Scores for lameness, swelling, and lesions were totaled to arrive at a footrot
score.
Biopsy Sampling of Subcutaneous Tissue: Tissue biopsy specimens were
obtained from each experimentally infected animal six days after inoculation
with
bacteria, and immediately prior to antibiotic therapy. Each animal had its
affected foot
physically restrained using ropes. The foot was lifted and cleaned using water
and
disinfectant solution followed by 70:30 (vol/vol) ethanol:water. Local
anaesthesia was
applied and a small incision was made using a scalpel blade (#10) over a
swollen
portion of the limb immediately proximal to the affected region of the foot. A
TrucutT'"
hepatic biopsy needle was inserted into the subcutaneous tissue. A core of
tissue was
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collected and transported immediately to the laboratory in anaerobic transport
medium.
A second biopsy specimen was collected from each animal and fixed in 10%
neutral
buffered formalin for histology.
Bacteriology: Tissue samples were placed in modified chopped meat medium
and incubated at 37°C under anaerobic conditions. Periodically, a small
sample of
broth was removed from the broth culture and streaked on kanamycin:vancomycin
faked blood agar, Brucella agar, and on chocolate agar to obtain single
colonies of the
anaerobic bacteria present. Isolates were presumptively identified based on
colony
morphology and pigmentation, fluorescence under longwave UV light, gram stain
reaction, cellular morphology, antibiotic resistance (kanamycin, vancomycin,
colistin},
catalase production, nitrate test, indole test, lipase production on egg yolk
agar, and the
urease and oxidase tests.
Antibiotic Sensitivity of Bacterial Isolates: Bacteria were grown
anaerobically
on kanamycin:vancomycin faked blood agar for colony isolation and purity.
Isolates
IS were then inoculated into 10 ml of modified chopped meat medium and
incubated
anaerobically at 37°C in an environmental chamber. Oxytetracycline
hydrochloride
and a macrolide antibiotic were, diluted in medium to a concentration of 640
~.g/ml.
Serial dilutions (1.1) were made for both compounds to yield stock solution
ranging
from 640 ug/ml to 0.0625 ug/ml. Falcon 3072 Micro Test IIIT"' tissue culture
plates
with lids were loaded in triplicate with 100 ~,1 of each concentrations (MIC).
Each drug
concentration was inoculated with 100 ~ul of bacterial broth inoculum. The
inoculum
diluted l:l in sterile PBS (pH 7.2) served as a control. Drug stock solution
was also
aliquoted to measure absorbance capabilities (650 nm). Plates were incubated
for 24h
anaerobically at 37°C. The broth inoculum was serially diluted, plated
on
kanamycin:vancomycin faked blood agar and incubated anaerobically for viable
counts
(CFU/ml). After the incubation period, 20 ul of inoculum-drug combination was
removed from one series of each descending drug concentration and plated on
kanamycin:vancomycin faked blood agar to determine minimum bactericidal
concentration (MBC) values. All MBC plates were incubated for seven days under
anaerobic conditions. The tissue culture plates were then read on a microplate
reader
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using SOFTmax Version 2.32 software. Mean absorbance (650 nm) readings and
grey
scale analysis were used to determine the MIC for each drug and isolate
tested. The
MBC was determined by the drug concentration which exhibited a 99.9 %
reduction of
CFU/ml count.
All six steers were normal and healthy prior to the study. No concurrent
disease occurred during the period of this study. All six steers developed a
clinical
condition consistent with acute footrot within five days following inoculation
of the
bacteria into the subcutaneous tissue and the skin of the interdigital cleft.
Each of the
six animals was also clinically lame by five days post-inoculation with
bacteria.
Biopsy specimens were successfully obtained from all animals on the sixth day
following experimental infection. In each case anaerobic bacteriology produced
the re-
isolation of the organisms initially injected into the feet. Microscopic
lesions were very
similar in all steers. Typical lesions involved coagulative necrosis and edema
of the
subcutaneous tissues and an infiltration of local tissue with monocytic
inflammatory
cells. Very few neutrophils were seen; however, biopsy samples were taken six
days
after exposure to the bacteria. An incubation period of five days after
inoculation with
bacteria may suggest that an initial failed neutrophilic response followed by
a more
chronic monocytic infiltration may precede clinical signs in this model
infection. The
implications of this finding in natural infections of footrot are not known.
Morphotypic
bacteria were visible in stained sections of the biopsy specimens.
EXAMPLE 6
Assessment of the Effectiveness of a Macrolide Antibiotic
to Treat Footrot
Treatment: Six days following experimental inoculation of feet with bacteria,
and immediately following biopsy specimen collection, all steers from Example
S were
treated with a single subcutaneous administration of a macrolide antibiotic at
a dosage
of 10 mg per kilogram body weight. Response to the therapy was monitored by
clinical
scoring, as outlined above. Twenty-four (24) hours following administration of
the
antibiotic, the feet were prepared for biopsy sampling of subcutaneous tissue
and a third
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biopsy taken, held on dry ice for transportation to the laboratory, frozen in
1~~,~, and
stored at -85 °C.
Histology: Tissue specimens fixed in 10% neutral buffered formalin were
dehydrated in solutions of increasing ethanol concentration followed by three
15 min
exposures to absolute ethanol. The specimens were then infiltrated with and
embedded
in meta-methacrylate. Sections (1.5 ~,m) were cut on a microtome, heat fixed
on
microscope slides, and stained with buffered methylene blue and basic fuchsin
(19].
Coverslips were fixed on slides with mounting medium and sections were viewed
and
photographed on a microscope.
Statistical Analysis: Clinical scores were evaluated by comparison to pre-
exposure scores in the same groups of animals using nonparametric analysis of
variance
and Kruskal Wallis tests. Data of Group 1 and Group 2 were compared by
nonparametric analysis of variance and Mann Whitney tests. In all cases the P
value
was 0.05. The mean total footrot scores for animals in Group 1 and Group 2 are
shown in Fig. 6A and Fig. 6B, respectively. In the Porphyromonas levii
infected
animals (Group 1) the mean total footrot score from day 7 was significantly
reduced
following antibiotic therapy on day 6. Scores remained significantly lower
than on day
6 and continued to decrease from day 8 to day 17. In the Prevotella intermedia
infected
animals (Group 2) there was a reduction in clinical score; however, the
reduction was
not statistically significant. Fig. 7 demonstrates the mean total footrot
scores from all
six animals (irrespective of group). There was significant improvement in the
footrot
score in five of the six animals following treatment and this reduction in
clinical score
was maintained for the entire 17 days of the experiment. The single failure
(animal
number YEL398) was initially the most severely affected animal following
experimental infection. It did not respond to a single administration of the
macrolide
antibiotic, or to a second administration of the drug 48h later. Treatment
with ceftiofur
sodium at a dosage of 1.0 mg ceftiofur per kg body weight q24h for five days
was
implemented following the initial treatment course. The steer retained a
subtle and
permanent lameness and was eventually euthanized 30 days following the last
treatment
and given a necropsy examination. The post mortem examination revealed
fibrosis of
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the lateral branch of the long digital extensor tendon. There was no evidence
of active
infection grossly or histologically. Fig. 8 shows the mean total footrot
scores of
animals excluding the non-responding YEL398.
Table 3 demonstrates the minimum inhibitory concentrations and minimum
S bactericidal concentrations of the three strains of bacteria used to
experimentally infect
feet. The MIC of the organisms re-isolated from infected feet immediately
prior to
administration of macrolide antibiotic did not differ substantially from those
in Table 3.
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Table 3
MIC and MBC of Macrolide Antibiotic
Bacteria MIC (p,g/ml) MBC (tcm/ml)
Porphyromonas levii < 0.03 > 32
Prevotella intermedia 4 32
Fusobacterium necrophorum 0.13 4
The macrolide antibiotic tested in the experimental model was effective in
treating five of six animals (83.3%) with experimentally-induced acute
footrot. This
occurred despite seemingly unfavorable MBC value for both Porphyromonas levii
and
Prevotella intermedia. This antibiotic is concentrated within monocytic cells,
such as
macrophages, at levels much higher than those found in serum and tissue. The
ability
of these tissue macrophages to kill bacteria may be accentuated in the
presence of
intracellular antibiotic and this may be a fundamental reason for the success
of the
antibiotic with bacteria having unfavorable MBC's. Although more data are
required to
fully establish the usefulness of this antibiotic in bovine footrot, it may be
an effective
antibiotic for use in this disease based on its performance in the
experimental model for
this disease.
EXAMPLE 7
Immunization of Cattle Study
General Design and Blood Sampling Times: A total of 26 individually
identified cattle were used for this study. They were randomly allocated to
one of the
following groups: 1] immunized or 2) sham-immunized. Prior to immunization
each
animal had a venous blood specimen collected from the jugular vein by standard
venipuncture technique (day 0). Serum was separated from this blood specimen
and
stored frozen at -85°C. Animals were immunized on day 1. Blood samples
for serum
were collected three weeks following initial immunization (day 21) when the
animals
received a second immunization (booster injection of identical preparation).
Seven days
following the booster injections (day 28) sera were collected again, and the
cattle were
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inoculated with viable Porphyromonas levii and Fusobacterium necrophorum, as
described in Example 5. Serum samples were collected again on day 35, day 42
and at
six months.
Vaccination: Cattle were immunized with: 1) an inactivated whole cell vaccine
composition (bacterin) composed of a snap-frozen: thawed preparation of
Prevotella
intermedia and Porphyromonas levii, as described above, and alum adjuvant or
2)
saline and alum adjuvant. The bacterin contained approximately 0.5 to 0.75 mg
protein
per ml and each animal received about 1 ml of bacterin containing about 0.75
mg of
protein prepared with commercially available alum-based adjuvant by a
subcutaneous
route. Animals were immunized or sham-immunized on day 1 and day 21. The
bacterins were assessed for IgG protease activity by the methods outlined in
Example 3
and for ~3-lactamase activity using nitrocefin discs, as described in Example
4. The P.
levii bacterin was found to have protease activity but no ~3-lactamase
activity. while the
P. intermedia bacterin was found to have (3-lactamase activity but no protease
activity.
Clinical Response: The individual animals were the experimental unit. Clinical
scores were assigned daily to animals, using the system described in Example
5, by an
individual blinded to experimental group. Cattle that were vaccinated had
lower
clinical scores than sham vaccinated controls during experimentally induced
infection.
This study demonstrated that pre-exposure immunization with Porpln~romonas
levii and Prevotella intermedia resulted in the production of serum antibody
that were
protective in experimental footrot infections. The serum antibody specifically
reacting
to Porphyromonas levii and Prevotella intermedia was more abundant in
immunized
animals than in sham-immunized animals. In particular, levels of serum IgG
specific
for P. levii were significantly higher than the levels in sham vaccinated
animals both
during and following experimental infection, i.e., between day 28 and 187. The
serum
antibodies reactive to Fusobacterium necrophorum were similar in groups of
immunized animals and sham-immunized animals during the entire duration of the
experiment (all time points sampled). The antibodies produced by animals
immunized
with our vaccine conveyed protection from severity of experimental infection
compared
to sham-immunized animals, as measured by clinical response and scoring. The
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severity of experimental infection within individual animals generally
correlated with
the serum antibody response in those individual animals.
EXAMPLE 8
Neutrophil-Mediated Phagocytosis and the Production of
Immunoglobulin GZ Protease by Porphyromonas Levii
Methods: PMN were purified from whole bovine blood, quantified, assessed
for viability, and exposed to one of three putative etiologic agents of AIP
(Porphyromonas levii, Prevotella intermedia, Fusobacterium necrophorum ATCC
27852) or latex beads and assessed for phagocytosis using direct microscopy.
The
effects of bovine serum specifically raised against P. levii on antibody-
mediated
phagocytosis by PMN (opsonization) were examined. P. levii was examined for
the
presence of protease activity capable of cleaving bovine Ig (IgG, IgM, IgG,,
IgG2).
Blood Collection and PMN Purification: Whole bovine blood was collected in
acid citrate dextrose by jugular venipuncture. The erythrocyte fraction was
separated
by centrifugation and this fraction subjected to repeated cold hypotonic lysis
[38] .
PMN purified in this fashion were pelleted by centrifugation, resuspended in
sterile
pyrogen-free PBS, enumerated by haemacytometry, viability determined by
staining
with 0.1 % trypan blue, and assessed for differential leukocyte population on
cytospin
preparations. Preparations were required to be a minimum 95 % pure and 95 %
viable
for assays to be performed.
Antibody Production and Evaluation: A calf with low pre-exposure titres to all
three bacterial antigen preparations was used. P. levii was grown in anaerobic
conditions, killed with 10% buffered formalin, washed three times, suspended
at 1.0
mg protein per mL, and mixed with alum adjuvant prior to immunization of the
calf.
The calf received a booster injection 21 days later with the appropriate
antigen
preparation. Antibody titres were determined by ELISA using killed whole cells
of P.
levii as the antigen preparation.
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Phagocytosis Assays: Purified viable PMN (1.0 x 10~ cells/mL) were incubated
for 15 min (37°C) with 1.5 x 10' bacteria/mL following pre-exposure of
bacteria to
either sterile pyrogen-free PBS, low titre serum, or high titre serum for 30
min. The
mixture was centrifuged to pellet PMN, the cells re-suspended in PBS, and the
solution
S cytospinned and stained with Diff-Quik stain for microscopic viewing. Counts
of PMN
cells phagocytosing bacteria per total cells were determined and photographs
were
taken.
Protease Preparation, Ig Biotinylation, and Ig Protease Assay: Supernatant of
a
broth culture of P. levii grown in anaerobic conditions (5% Ii2: 5% COz:
90%NZ) in
supplemented cooked meat broth (haemin, Vitamin K) at 37°C for four
days was
collected by centrifugation and stored (-80°C) until used in the
protease assay. Culture
purity was assured by streak plating onto BBA and standard bacteriological
identification.
N-hydroxy-succinomidobiotin was dissolved in dimethyl sulfoxide and was
added to each of bovine IgG, IgM, IgG, and IgG2 (1:10 wt/vol), the Igs were
incubated
for 2h (25 °C), and the reaction terminated with 1 mg of glycine. The
biotinylated Igs
were dialyzed 14h in three changes of double distilled water and final protein
concentrations were determined by Bradford assay [39] .
The protease assay was a slight modification of the methods of Lee and Shewen
[24]. Biotinylated IgG (10 fig) was added to 250 ~,L of supernatant and 2.5
~,L of
penicillin-streptomycin solution (penicillin G 100 U/mL, streptomycin 100
~cg/mL).
The protease assay was carried out at 30, 35 and 40°C and optimal assay
temperature
was determined to be 35°C. Samples (10 ~,L) were taken at 0, 0.5, 1, 2,
4, 8, 24, 48,
and 72h and added to 10 ~.L of SDS PAGE sample buffer [30], boiled for 4 min,
followed by electrophoretic separation by SDS PAGE. Control assays included P.
levii
supernatant heated to 100°C prior to exposure to the biotinylated Igs
as well as
uninoculated cooked meat medium and distilled water.
All samples were resolved in 15% SDS PAGE gels using a 4% stacking gel and
standard methodology. Gels were run on ice at 150V for 15 min followed by 200V
for
45 min in 10% running buffer. Protein in the gels was transferred to
nitrocellulose [31]
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at 20V for 14h at 4°C, washed in blocking solution (lh at 37°C),
washed with TBS-
tween buffer (3 x 15 min 20°C), and incubated with streptavidin-
alkaline phosphatase
(1:5000) in TBS-tween for 2h at 37°C. Blots were again washed three
times for 15 min
in TBS-tween and nitroblue tetrazolium (NBT) and 5-bromo- 4 chloro- 3 indolyl
phosphate (BCIP) were added as substrate for color development (4 min) in
buffer
(31.52 g Tris-HCL, 0.18 g MgCl2-6H.,0, 1 1 distilled water, pH 8.8). BCIP was
initially dissolved in DMSO. Protein bands representing the light and heavy
chains of
the biotinylated immunoglobulins, or fragments of these immunoglobulin chains,
were
viewed based on alkaline phosphatase activity on the nitrocellulose.
Photographs were
obtained immediately.
Results of Phagocytosis Assays: Comparisons of the ability of bovine PMN to
phagocytose each of the putative etiologic agents of AIP are shown in Table 4.
Values
are expressed as mean cells phagocytosing bacteria per 100 PMN ~ SD (n value
is 9
per data point). * = significantly different than all other experimental
groups
(P < 0.05). Bovine PMN readily phagocytose P. intermedia and F. necrophorum,
but
the ability of the cells to phagocytose P. levii was significantly reduced
compared to
these groups and to PBS controls (P< 0.05).
Table 4. Comparison of Phagocytosis by Bovine PMN in vitro
of Latex Beads, P. levii, P. intermedia and F. necrophorum
Experimental Group Phagocytosis
Latex beads 17.9 ~3.8
P. levii 5.3 t 1.6*
P. intermedia (8-2) 60.7 ~ 6.1
F. necrophorum 64.1 t 4.4
The phagocytic abilities of PMN for P. levii when the bacteria were pre-
incubated
with PBS, low titre anti-P. levii serum, high titre anti-P. levii serum, or
complement-
destroyed high titre anti-P. levii serum were examined. High levels of
phagocytosis were
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evident in these same cell preparations if the P. levii were pre-incubated
with high titre
anti-P. levii bovine serum. This restoration of phagocytic function was not
altered
substantially by heating the serum at 56°C for 30 min (Table 5). * =
significantly greater
than both PBS control and low titre serum (P< 0.05) but not different from
complement
destroyed serum.
Table 5. Examination of Phagocytosis Abilities of PMN for
P. levii when Incubated with Anti-P. levii Serum
Experimental Group Phagocytosis
P. levii and PBS 5.3 ~ 1.6
P. levii and low Ig 30.0 t 5.5
P. levii and high Ig 97.0 ~ 1.1 *
P. levii and inact. complement factors 89.9 ~ 3.8*
Ig Protease Assays: Nitrocellulose transfer of biotinylated IgG exposed to
culture supernatant of P. levii is shown in Fig. 9A. From left to right are
increasing times
(hours) of exposure. Lane A = 0, Lane B = 0.5, Lane C = 1, Lane D = 2, Lane E
= 4,
Lane F = 8, Lane G = 24, Lane H = 48, and Lane I = 72h time samples. Results
show the
presence and increasing intensity of the degradation bands (major bands are
highlighted
with arrows) smaller than the heavy and light chains (arrowheads) as
incubation time
increases. Also, near complete degradation of the heavy chain by 24h was
noted.
In contrast, nitrocellulose transfer of biotinylated IgM exposed to culture
supernatant of P. levii is shown in Fig. 9B. From left to right are increasing
times (hours)
of exposure. Lane A = 0, Lane B = 0.5, Lane C = 1, Lane D = 4, Lane E = 8,
Lane F =
24, Lane G = 48, and Lane H = 72h time samples. There is absence of
degradation
bands. Only the light and heavy chains (arrowheads) of IgM are evident.
When IgG, was assayed, degradation bands were not evident (Fig. l0A) and again
only bands representing the heavy and light Ig chains could be seen. However,
when
assays using IgG, were conducted similar results to those employing crude IgG
were seen
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CA 02300958 2000-02-17
WO 99/12564 PCT/CA98/00823
with degradation bands appearing within 30 min, as illustrated in Fig. l OB,
but heavy
chain was not as extensively degraded. Controls using water alone, heated
supernatant,
supernatant extracts of P. intermed~a or F. necrophorum, or uninoculated media
(each of
which was exposed to the biotinylated IgG) showed no evidence of degradation
bands
S (data not shown).
Modification of the above-described modes of carrying out the various
embodiments of this invention will be apparent to those skilled in the art
following the
teachings of this invention as set forth herein. The examples described above
are not
limiting, but are merely exemplary of this invention, the scope of which is
defined by
the claims which follow.
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