U.S. patent application number 14/027385 was filed with the patent office on 2014-01-09 for actinobacillus suis antigens.
This patent application is currently assigned to BOEHRINGER INGELHEIM VETMEDICA, INC.. The applicant listed for this patent is Boehringer Ingelheim Vetmedica, Inc.. Invention is credited to Dianna M. Murphy JORDAN, Jeremy J. KROLL, Philip UTLEY.
Application Number | 20140011210 14/027385 |
Document ID | / |
Family ID | 43428689 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011210 |
Kind Code |
A1 |
KROLL; Jeremy J. ; et
al. |
January 9, 2014 |
ACTINOBACILLUS SUIS ANTIGENS
Abstract
The invention provides immunogenic compositions useful for
inhibiting, treating, protecting, or preventing infection by
Actinobacillus suis. These immunogenic compositions are
demonstrated to usefully stimulate immunogenic responses in treated
pigs. Some vaccines stimulated reactions sufficient to be
protective against A. suis. In addition, the invention provides
kits comprising the immunogenic compositions; as well as, methods
of using the compositions and kits.
Inventors: |
KROLL; Jeremy J.;
(Urbandale, IA) ; UTLEY; Philip; (Slater, IA)
; JORDAN; Dianna M. Murphy; (Ames, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boehringer Ingelheim Vetmedica, Inc. |
St. Joseph |
MO |
US |
|
|
Assignee: |
BOEHRINGER INGELHEIM VETMEDICA,
INC.
St. Joseph
MO
|
Family ID: |
43428689 |
Appl. No.: |
14/027385 |
Filed: |
September 16, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12942579 |
Nov 9, 2010 |
8563005 |
|
|
14027385 |
|
|
|
|
61259728 |
Nov 10, 2009 |
|
|
|
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 2039/552 20130101; A61P 37/04 20180101; G01N 33/56911
20130101; A61K 39/102 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A method of diagnosing Actinobacillus suis infection in a
subject, comprising a) providing a filtered supernatant prepared by
growing an A. suis culture to between 0.650 OD.sub.650 and 0.850
OD.sub.650, collecting supernatant from the culture, and filtering
the supernatant; b) contacting the filtered supernatant with a
sample obtained from the subject; and c) identifying the subject as
having an A. suis infection if an antibody capable of binding a
component in the filtered supernatant is detected in the
sample.
2. The method of claim 1, wherein the binding is detected using a
second antibody capable of binding the antibody in the sample.
Description
BACKGROUND OF THE INVENTION
[0001] A. Field of the Invention
[0002] The present invention relates to methods and compositions
useful for inhibiting, treating, protecting, or preventing
infection by Actinobacillus suis.
[0003] B. Description of the Related Art
[0004] Actinobacillus suis has recently emerged as a new threat to
the swine industry in the United States. Previously associated with
high mortality in "high health" herds, A. suis is now recognized as
an important pathogen of conventional herds. It is particularly
detrimental to younger animals. Infection results in
actinobacillosis and can cause sudden death in both neonate and
weaned pigs. Symptoms in weaned pigs include anorexia, fever,
cyanosis, congestion of the extremities, respiratory distress,
pneumonia, necrotizing pneumonia, persistent cough, skin lesions,
and fatal septicemia. Pneumonia, arthritis, septicemic signs,
pleurisy, pericarditis, and miliary abscesses are known to occur in
finishing pigs. Actinobacillosis also causes metritis and abortion
in sows.
[0005] The gross pathology of actinobacillosis is characterized by
lesions found in lungs, kidney, heart, liver, spleen, intestines
and skin; hemorrhages and necrosis; and pneumonic lesions
resembling pleuropneumonia. Histopathologically, the disease is
characterized by the presence of bacterial thromboemboli with
accompanying fibrinohemorrhagic necrosis in the vessels of various
tissues; necrotizing bronchopneumonia; and pleuritis. Causes and
contributing factors to infection include precipitation by Porcine
Reproductive and Respiratory Syndrome (PRRS) infection, teeth
clipping, de-tailing, scrubbed knees, and entry via either
respiration, cuts, or abrasions.
[0006] Actinobacillus suis is an opportunistic, gram-negative,
non-motile, aerobic and facultative anaerobic coccobacillus that
colonizes the upper respiratory tract. Genotyping of A. suis
isolates recovered from clinical cases in the North American swine
herds has revealed a limited genetic variability, with only 13
strains being identified among 74 isolates recovered from 29
different herds. The Simpson's diversity index (also known as
species diversity index, see Simpson, 1949) for A. suis genotypes
is 0.64, meaning that a random isolate has a 64% chance of being
included in a unique genotype group using for example BOX-PCR
(Simpson, 1949; Versalovic et al, 1991; Oliveira et al, 2007).
Compared with H. parasuis, for example, which has a diversity index
of 0.93 (Oliveira et al, 2007), A. suis is relatively clonal.
[0007] The phenotypic diversity of A. suis is also relatively
limited. Only 2 serovars, namely O1 and O2 (Rullo, Papp-Szabo and
Michael, 2006), and three capsular types, K1-3, have been described
so far. Pathogenicity studies suggest that isolates from serogroup
O2 tend to be more virulent than O1 isolates (Slavic, DeLay and
Hayes, 2000). Serotyping of A. suis isolates used for autogenous
vaccine production also confirms that a higher percentage of O2
isolates were associated with clinical disease compared with O1
isolates (Slavic, Toffner, and Monteiro, 2000). Although some of
the A. suis virulence factors are known (e.g. the RTX toxins Apx
I.sub.var. suis and Apx II.sub.var. suis), the factors that may
trigger systemic infection still remain to be defined. Some of
these potential factors include lipopolysaccharide (LPS) and
capsular polysaccharides (CPS), outer membrane protein A (OmpA),
proteases, and iron acquisition.
[0008] Currently, there are no commercial vaccines available for
the control of A. suis, and most field veterinarians rely on
autogenous vaccines and antimicrobial treatments to control
disease. The development of a vaccine that will potentially protect
against most isolates in the field is desirable; however, necessary
data regarding the association between genotype, serovar, toxin,
protein profiles, and factors that are involved in the pathogenesis
of A. suis infection still remain to be defined. Herein, such data
are provided, as well as, vaccines, and their methods of use,
against A. suis.
SUMMARY OF THE INVENTION
[0009] The present invention provides new immunogenic compositions
that are useful for protecting a subject against Actinobacillus
suis infection. These compositions are also useful for inhibiting,
treating, or preventing infection by various strains or types of
Actinobacillus suis.
[0010] Compositions of the invention comprise a supernatant
collected from one or more A. suis cultures grown to between 0.650
OD.sub.650 and 0.850 OD.sub.650; and an adjuvant. Preferably the
supernatant is inactivated, most preferably by formalin. The
supernatant is also preferably filtered, such as through a 45
micron filter. Filtration may occur before or after inactivation as
deemed appropriate for a given situation. The supernatant, more
preferably the filtered supernatant, is essentially free from A.
suis cells but may contain multiple cellular components.
[0011] The skilled artisan will recognize that any of a variety of
adjuvants may be suitably included in a composition of the
invention. One exemplary adjuvant is Emulsigen.RTM.-D. The
determination of adjuvant will, in part, depend upon the nature of
the subject that is to receive the composition; the method of
administration to the subject; and conditions under which the
composition is to be administered. For example, the adjuvant and
supernatant, or filtered supernatant, may be admixed together prior
to administration to a subject, administered simultaneously, or
administered sequentially to a subject.
[0012] Suitable subjects of the immunogenic compositions of the
invention include animals and humans. Animals in which the immune
response is stimulated by use of compositions or methods of the
invention include livestock, such as pigs, calves, chickens, goats,
and sheep, and domestic animals, such as mice, rabbits, dogs, cats,
and horses. Preferred animals include porcines, murids, equids,
lagomorphs, and bovids. Most preferably the animal is a
porcine.
[0013] The invention also provides methods of provoking an immune
response against Actinobacillus reducing the incidence of or
severity of a clinical sign associated with Actinobacillus suis
infection in a subject comprising administering to the subject an
immunogenic composition of the invention. Clinical signs associated
with A. suis that may be reduced in incidence or severity in a
subject include meningitis, septicemia, metritis, pneumonia,
crysipelas-like lesions, and abortion. Any one of which may be
lessened by the administration of a composition of the invention
relative to a subject not receiving the immunogenic composition.
Preferred compositions of the invention elicit a protective
immunological response that is at least a 10% reduction in at least
one clinical sign of an A. suis infection.
[0014] A preferred Actinobacillus suis infection that may be
reduced by administration of a composition of the invention is A.
suis ISU-8594.
[0015] The invention also provides methods of making or preparing
immunogenic compositions of the invention that may be useful in the
making of a medicament. Such methods include the steps of growing
an Actinobacillus suis culture to between 0.650 OD.sub.650 and
0.850 OD.sub.650; collecting a supernatant from the culture;
filtering the supernatant to yield a filtrate; and mixing the
filtrate with an adjuvant. Such methods may also include
inactivating the supernatant, preferably prior to admixing the
supernatant with an adjuvant. Inactivation may occur either before
or after filtering the supernatant.
[0016] The invention further provides methods of diagnosing an
Actinobacillus suis infection in a subject. Such methods comprise:
a) providing a filtered supernatant prepared by growing an A. suis
culture to between 0.650 OD.sub.650 and 0.850 OD.sub.650,
collecting supernatant from the culture, and filtering the
supernatant; b) contacting the filtered supernatant with a sample
obtained from the subject; and c) identifying the subject as having
an A. suis infection if an antibody capable of binding a component
in the filtered supernatant is detected in the sample.
[0017] Those of skill in the art will be familiar with a variety of
techniques suitable for ascertaining if an antibody is capable of
binding to a component. For example, binding may be detected by
using a second antibody capable of binding the antibody in the
sample. Binding by such second antibodies may by detected by a
colorimetric assay or other suitable means.
[0018] The invention also provides kits that comprise: a) a
filtered supernatant prepared by growing an Actinobacillus suis
culture to between 0.650 OD.sub.650 and 0.850 OD.sub.650,
collecting a supernatant of the culture, and filtering the
supernatant; b) an adjuvant; and c) a container for packaging the
supernatant and adjuvant. The filtered supernatant and adjuvant may
be packaged together or separately.
[0019] A kit may further comprise instructions for use of the kit.
It may also comprise a means of administering the filtered
supernatant and adjuvant to a subject. A means of admixing the
supernatant and adjuvant together may also be included in a
kit.
[0020] Compositions of the invention may further comprise a
veterinarily acceptable carrier, second adjuvant, or combination
thereof. Such compositions may be used as a vaccine and comprise an
attenuated vaccine, an inactivated vaccine, or combinations
thereof. Such vaccines elicit a protective immunological response
against at least one disease associated with Actinobacillus.
[0021] Preferred inactivation agents for use in methods of the
invention are selected from the group consisting of binary
ethyleneimine (BEI) and formalin. Formalin is a more preferred
inactivation agent. Those of skill in the art will recognize that
other inactivation agents and methods (i.e. heating, changing pH,
etc.) are known in the art and may be interchangeably used in the
practice of the invention, as long as, such agents or deactivation
methods do not adversely alter the immunogenic properties or safety
of the composition produced.
[0022] Methods of the invention may also comprise admixing a
composition of the invention with a veterinarily acceptable
carrier, adjuvant, or combination thereof. Those of skill in the
art will recognize that the choice of carrier, adjuvant, or
combination will be determined by the delivery route, personal
preference, and animal species among others.
[0023] Preferred routes of administration include intranasal, oral,
intradermal, and intramuscular. Administration in drinking water,
most preferably in a single dose, is preferred. The skilled artisan
will recognize that compositions of the invention may also be
administered in two or more doses, as well as, by other routes of
administration. For example, such other routes include
subcutaneously, intracutaneously, intravenously, intravascularly,
intraarterially, intraperitnoeally, intrathecally, intratracheally,
intracutaneously, intracardially, intralobally, intramedullarly,
intrapulmonarily, or intravaginally. Depending on the desired
duration and effectiveness of the treatment, the compositions
according to the invention may be administered once or several
times, also intermittently, for instance on a daily basis for
several days, weeks or months and in different dosages.
[0024] The invention also provides kits for vaccinating a subject
comprising a set of printed instructions; a dispenser capable of
administering a vaccine to an animal; and a supernatant from an A.
suis culture having one or more components that effectively
stimulates an immune response in a subject. Kits of the invention
may further comprise a veterinarily acceptable carrier, adjuvant,
or combination thereof.
[0025] A dispenser in a kit of the invention is capable of
dispensing its contents as droplets; and the A. suis supernatant
included in the kit is capable of reducing the severity of at least
one clinical sign of an A. suis infection when administered to a
subject. Preferably, the severity of a clinical sign is reduced by
at least 10% as compared to an untreated, infected subject.
[0026] An "immunogenic or immunological composition" refers to a
composition of matter that comprises at least one A. suis
supernatant, or immunogenic portion thereof, that elicits an
immunological response of a cellular or antibody-mediated immune
response to the composition in the subject. In a preferred
embodiment of the present invention, an immunogenic composition
induces an immune response and, more preferably, confers protective
immunity against one or more of the clinical signs of a
Actinobacillus infection.
[0027] An "immune response" or "immunological response" means, but
is not limited to, the development of a cellular and/or
antibody-mediated immune response to the composition or vaccine of
interest. Usually, an immune or immunological response includes,
but is not limited to, one or more of the following effects: the
production or activation of 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. Preferably, the vaccinated subject will display either a
therapeutic or a protective immunological (memory) response such
that resistance to new infection will be enhanced and/or the
clinical severity of the disease reduced. Such protection will be
demonstrated by either a reduction in number of symptoms, severity
of symptoms, or the lack of one or more of the symptoms associated
with the infection of the pathogen, a delay in the of onset of
clinical symptoms, reduced pathogen persistence, a reduction in the
overall pathogen load and/or a reduction of pathogen excretion.
[0028] "Protection against A. suis", "protective immunity",
"functional immunity", and similar phrases, mean an immune response
against A. suis generated by an immunization schedule that results
in fewer deleterious effects than would be expected in a
non-immunized subject that has not been previously exposed to A.
suis. That is, the severity of the deleterious effects of the
infection are lessened in an immunized subject because the
subject's immune system is resistant to the bacterium. Infection
may be reduced, slowed, or possibly fully prevented, in an
immunized subject, preferably a pig. Herein, where complete
prevention of infection is meant, it is specifically stated. If
complete prevention is not stated then the term includes partial
prevention.
[0029] Herein, "reduction of the incidence and/or severity of
clinical signs" or "reduction of clinical symptoms" means, but is
not limited to, reducing the number of infected subjects in a
group, reducing or eliminating the number of subjects exhibiting
clinical signs of infection, or reducing the severity of any
clinical signs that are present in the subjects, in comparison to
wild-type infection. For example, it should refer to any reduction
of pathogen load, pathogen shedding, reduction in pathogen
transmission, or reduction of any clinical sign symptomatic of A.
suis infection. Preferably these clinical signs are reduced in
subjects receiving the composition of the present invention by at
least 10% in comparison to subjects not receiving the composition
and may become infected. More preferably clinical signs are reduced
in subjects receiving the composition of the present invention by
at least 20%, preferably by at least 30%, more preferably by at
least 40%, and even more preferably by at least 50%.
[0030] The term "increased protection" herein means, but is not
limited to, a statistically significant reduction of one or more
clinical symptoms which are associated with A. suis infection in a
vaccinated group of subjects vs. a non-vaccinated control group of
subjects. The term "statistically significant reduction of clinical
symptoms" means, but is not limited to, the frequency in the
incidence of at least one clinical symptom in the vaccinated group
of subjects is at least 20%, preferably 30%, more preferably 50%,
and even more preferably 70% lower than in the non-vaccinated
control group after the challenge with an infectious Actinobacillus
bacterium.
[0031] Those of skill in the art will understand that the
compositions used herein may incorporate known injectable,
physiologically acceptable sterile solutions. For preparing a
ready-to-use solution for parenteral injection or infusion, aqueous
isotonic solutions, e.g. saline or plasma protein solutions, are
readily available. In addition, the immunogenic and vaccine
compositions of the present invention can include
veterinary-acceptable carriers, diluents, isotonic agents,
stabilizers, or adjuvants.
[0032] As used herein, "a veterinary-acceptable carrier" includes
any and all solvents, dispersion media, coatings, adjuvants,
stabilizing agents, diluents, preservatives, antibacterial and
antifungal agents, isotonic agents, adsorption delaying agents, and
the like. In some preferred embodiments, and especially those that
include lyophilized immunogenic compositions, stabilizing agents
for use in the present invention include stabilizers for
lyophilization or freeze-drying.
[0033] In some embodiments, the immunogenic composition of the
present invention contains an adjuvant. "Adjuvants" as used herein,
can include aluminum hydroxide and aluminum phosphate, saponins
e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.),
GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.),
water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water
emulsion. The emulsion can be based in particular on light liquid
paraffin oil (European Pharmacopea type); isoprenoid oil such as
squalane or squalene; oil resulting from the oligomerization of
alkenes, in particular of isobutene or decene; esters of acids or
of alcohols containing a linear alkyl group, more particularly
plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate),
glyceryl tri-(caprylate/caprate) or propylene glycol dioleate;
esters of branched fatty acids or alcohols, in particular
isostearic acid esters. The oil is used in combination with
emulsifiers to form the emulsion. The emulsifiers are preferably
nonionic surfactants, in particular esters of sorbitan, of mannide
(e.g. anhydromannitol oleate), of glycol, of polyglycerol, of
propylene glycol and of oleic, isostearic, ricinoleic or
hydroxystearic acid, which are optionally ethoxylated, and
polyoxypropylene-polyoxyethylene copolymer blocks, in particular
the Pluronic products, especially L121. See Hunter et al., The
Theory and Practical Application of Adjuvants (Ed Stewart-Tull, D.
E. S.). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al.,
Vaccine 15:564-570 (1997).
[0034] Other exemplary adjuvants are the SPT emulsion described on
page 147 of "Vaccine Design, The Subunit and Adjuvant Approach"
edited by M. Powell and M. Newman, Plenum Press, 1995, and the
emulsion MF59 described on page 183 of this same book.
[0035] A further instance of an adjuvant is a compound chosen from
the polymers of acrylic or methacrylic acid and the copolymers of
maleic anhydride and alkenyl derivative. Advantageous adjuvant
compounds are the polymers of acrylic or methacrylic acid which are
cross-linked, especially with polyalkenyl ethers of sugars or
polyalcohols. These compounds are known by the term carbomer
(Pharmeuropa Vol. 8, No. 2, June 1996). Persons skilled in the art
can also refer to U.S. Pat. No. 2,909,462 which describes such
acrylic polymers cross-linked with a polyhydroxylated compound
having at least 3 hydroxyl groups, preferably not more than 8, the
hydrogen atoms of at least three hydroxyls being replaced by
unsaturated aliphatic radicals having at least 2 carbon atoms. The
preferred radicals are those containing from 2 to 4 carbon atoms,
e.g. vinyls, allyls and other ethylenically unsaturated groups. The
unsaturated radicals may themselves contain other substituents,
such as methyl. The products sold under the name Carbopol; (BF
Goodrich, Ohio, USA) are particularly appropriate. They are
cross-linked with an allyl sucrose or with allyl pentaerythritol.
Among then, there may be mentioned Carbopol 974P, 934P and 971P.
Most preferred is the use of Carbopol 971P. Among the copolymers of
maleic anhydride and alkenyl derivative, are the copolymers EMA
(Monsanto), which are copolymers of maleic anhydride and ethylene.
The dissolution of these polymers in water leads to an acid
solution that will be neutralized, preferably to physiological pH,
in order to give the adjuvant solution into which the immunogenic,
immunological or vaccine composition itself will be
incorporated.
[0036] Further suitable adjuvants include, but are not limited to,
the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx,
Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl
lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin
from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or
muramyl dipeptide, or naturally occurring or recombinant cytokines
or analogs thereof or stimulants of endogenous cytokine release,
among many others.
[0037] "Diluents" can include water, saline, dextrose, ethanol,
glycerol, and the like. Isotonic agents can include sodium
chloride, dextrose, mannitol, sorbitol, and lactose, among others.
Stabilizers include albumin and alkali salts of
ethylendiamintetracetic acid, among others.
[0038] It is expected that an adjuvant can be added in an amount of
about 100 .mu.g to about 10 mg per dose, preferably in an amount of
about 100 .mu.g to about 10 mg per dose, more preferably in an
amount of about 500 .mu.g to about 5 mg per dose, even more
preferably in an amount of about 750 .mu.g to about 2.5 mg per
dose, and most preferably in an amount of about 1 mg per dose.
Alternatively, the adjuvant may be at a concentration of about 0.01
to 50%, preferably at a concentration of about 2% to 30%, more
preferably at a concentration of about 5% to 25%, still more
preferably at a concentration of about 7% to 22%, and most
preferably at a concentration of 10% to 20% by volume of the final
product.
[0039] Herein, "effective dose" means, but is not limited to, an
amount of A. suis supernatant or filtered supernatant that elicits,
or is able to elicit, an immune response that yields a reduction of
clinical symptoms in a subject to which the supernatant is
administered.
[0040] "Safety" refers to the absence of adverse consequences in a
vaccinated subject following vaccination, including but not limited
to: potential reversion of a bacterium-based vaccine to virulence,
clinically significant side effects such as persistent, systemic
illness or unacceptable inflammation at the site of vaccine
administration.
[0041] The terms "vaccination" or "vaccinating" or variants
thereof, as used herein means, but is not limited to, a process
which includes the administration of a composition of the invention
that, when administered to a subject, elicits, or is able to
elicit--directly or indirectly--an immune response in the subject
against A. suis.
[0042] Methods for the treatment or prophylaxis of infections
caused by A. suis are also disclosed. The method comprises
administering an effective amount of the immunogenic composition of
the present invention to a subject, wherein said treatment or
prophylaxis is selected from the group consisting of reducing signs
of A. suis infection, reducing the severity of or incidence of
clinical signs of A. suis infection, reducing the mortality of
subjects from A. suis infection, and combinations thereof.
[0043] "Mortality", in the context of the present invention, refers
to death caused by A. suis infection, and includes the situation
where the infection is so severe that an animal is euthanized to
prevent suffering and provide a humane ending to their life.
[0044] "Attenuation" means reducing the virulence of a pathogen. In
the present invention "attenuation" is synonymous with "avirulent".
In the present invention, an attenuated bacterium is one in which
the virulence has been reduced so that it does not cause clinical
signs of an A. suis infection but is capable of inducing an immune
response in the target subject, but may also mean that the clinical
signs are reduced in incidence or severity in subjects infected
with the attenuated A. suis in comparison with a "control group" of
subjects infected with non-attenuated A. suis and not receiving the
attenuated bacterium. In this context, the term "reduce/reduced"
means a reduction of at least 10%, preferably 25%, even more
preferably 50%, still more preferably 60%, even more preferably
70%, still more preferably 80%, even more preferably 90% and most
preferably of 100% as compared to the control group as defined
above.
[0045] An "effective amount" for purposes of the present invention,
means an amount of an immunogenic composition capable of inducing
an immune response that reduces the incidence of or lessens the
severity of A. suis infection in a subject. An effective amount
refers to colony forming units (CFU) per dose or logs/dose.
[0046] "Long-lasting protection" shall refer to "improved efficacy"
that persists for at least 3 weeks, but more preferably at least 3
months, still more preferably at least 6 months. It is most
preferred that the long lasting protection shall persist until the
average age at which porcine animals are marketed for meat.
[0047] Herein, unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular. In this application, the use of "or" means "and/or"
unless stated otherwise.
[0048] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0050] FIG. 1. Daily average temperatures among treatment groups
from day 0 to day 41 of the study.
DETAILED DESCRIPTION
[0051] The invention provides methods and compositions useful for
inhibiting, treating, protecting, or preventing infection by
Actinobacillus suis. Herein are described the effectiveness of A.
suis prototype vaccines against a heterologous challenge of various
serotypes or isolates of A. suis. These vaccines were demonstrated
to usefully stimulate immunogenic reactions in treated animals.
Some vaccines stimulated reactions sufficient to be protective
against A. suis.
[0052] The A. suis vaccine prototypes were comprised of different,
culture fractions (e.g. whole cell, supernatant, or outer membrane
protein (omp)) and a INGELVAC.RTM. APP-ALC live vaccine against A.
pleuropneumoniae (APP). The whole cell vaccine was made from the
pelleted material that resulted from a centrifugal spin of a cell
culture. The cultural supernatant was made from the supernatant
that resulted from the centrifugal spin, and may have included
exotoxins, endotoxins, secreted or sloughed-off omps, and other
cellular components that were not removed during the spin (see
Example 1). The INGELVAC.RTM. APP-ALC live vaccine was included
because APP encodes Apx I and II toxins that are very similar to A.
suis toxins. It was hypothesized that the INGELVAC.RTM. APP-ALC
vaccine may provide some cross protection against A. suis.
[0053] This efficacy study consisted of six treatment groups of
weaned 3 week-old.+-.7 days of age pigs. Treatment group 1 (15
pigs) received a 1.times.2 mL intramuscular (IM) dose of a
formalin-inactivated, whole cell fraction of A. suis on days 0 and
21 of the study, respectively. Treatment group 2 (15 pigs) received
a 1.times.2 mL 1M dose of a formalin-inactivated A. suis culture
supernatant fraction on days 0 and 21 of the study, respectively.
Treatment group 3 (15 pigs) received a 1.times.2 mL 1M dose of an
A. suis outer membrane protein (omp) cellular fraction on days 0
and 21 respectively. Treatment group 4 (15 pigs) received a
1.times.2 mL 1M dose of INGELVAC.RTM. APP-ALC on days 0 and 21
respectively. Treatment group 5 (15 pigs) designated as "challenge
controls" received a 1.times.2 mL IM dose of placebo on days 0 and
21 respectively. Treatment group 6 (10 pigs) were designated
"strict controls" and did not receive vaccine or placebo
treatment.
[0054] On the day of challenge (day 35) the pigs in groups 1-5
received a 6 mL dose/pig, 3 mL applied to each nostril, of A. suis
strain ISU-8594 via intranasal (IN) inoculation containing
1.times.10.sup.9.0 logs/dose. General observations were monitored
throughout the study, days 0 to 41, to record the overall health of
the pigs, as well as, injection site reactivity and rectal
temperatures. On Day 41 of the study, all animals were euthanized,
and all sections of the lung were scored for determining the
percentage of lung pathology. Fresh and fixed (lungs only) samples
of the lung (3 lobes with lesions, if present), liver, kidney,
spleen, tonsil, and swabs of nasal turbinates, trachea, bronchi,
meninges, and heart blood were collected from each animal and were
cultured for bacterial isolation and histopathology (lungs
only).
[0055] The A. suis fractions and placebo groups were adjuvanted
with Emulsigen.RTM.-D, and a few adverse injection site reactions
were noted in these groups. One pig in the supernatant group (group
2) was noted with a mild reaction at the injection site during the
first vaccination event. Overall, the Emulsigen.RTM.-D in
combination with the test articles did not produce any significant
injection site reactions in the supernatant and omp groups.
However, the whole cell group did have a few animals with abscesses
present at the time of necropsy, which could have been due to the
test article formulation that consisted of a concentrated whole
cell stock. The administration of these vaccine prototypes (i.e.
whole cell, supernatant, and omp) appeared to be well-tolerated
during the time of administration. The APP-ALC group had some
injection site swelling, which is consistent with the product
literature that notes swelling in 11% of the vaccinated
animals.
[0056] The general overall health of the pigs in each of the
treatment groups was good during the pre-challenge period. Only one
pig, from treatment group 4, was removed from the study during this
period. This pig was removed due to a bacterial infection believed
not to be associated with administration of the APP vaccine.
Further observations showed one pig in the challenge control group
was lame on its right leg from day 7 to day 24 of the study. This
pig skewed the clinical observation results during the
pre-challenge period and could have been considered an outlier and
removed from the study data analysis. Also, one pig showed poor
body condition in the whole cell group and died after a few days.
None of the pigs in the pre-vaccination period showed signs of
respiratory difficulties.
[0057] Assessment of the primary and secondary parameters were
validated by significant (p.ltoreq.0.05) increases in lesion
development that occurred in the challenge control group (treatment
group 5) compared to the strict control group (treatment group 6).
Results of this assessment validated the virulent pure culture A.
suis challenge model in pigs. Further comparisons were made against
the challenge control and not to the strict control group when
comparing groups 1-4.
[0058] During the six day challenge period one pig died of acute
death due to A. suis infection one day post-challenge in the whole
cell group. Based on previous challenge studies, the intranasal
route and the potency were appropriate to cause significant lesion
development in the test animals and apparent fatal sudden death, as
was the case for this one pig in the whole cell group.
[0059] Based on average lung lesion scores and percent with a
lesion, gross lung lesion scores appeared to be most severe in the
challenge group. Statistical analyses showed no statistical
differences (p.ltoreq.0.05) in average lung lesion scores or
percent with a lesion present when comparing groups 1 through 5.
Numerically the challenge controls received overall the highest
average gross lung lesion scores followed by the whole cell and omp
groups, which also received relatively high scores. Lesion
development was lower in the supernatant and APP groups, which
received lower numerical scores compared to the other groups. All
groups 1-5 had at least 50% of the pigs with a gross lesion score,
and the challenge and omp groups had as much as 80% of the pigs
with lesion development.
[0060] Furthermore, microscopic lung lesion analysis was based on
the severity of the hallmark traits of an A. suis infection, i.e.
bronchopneumonia, necrosis, and pleuritis. Bronchopneumonia and
necrosis were more severe in the challenge control and omp groups
where microscopic lesion scores were lower than in the supernatant
and APP groups. The APP group showed the best signs of protection
against an A. suis infection based on the microscopic lesion data
with statistical differences (p.ltoreq.0.05) found between group 3
(omp) for necrosis and pleuritis and the group 5 (challenge) for
pleuritis. Presence of bacterial colonies in all of the groups
ranged from about 20% to about 46.7% with the lowest percent
colonization occurring in the whole cell and APP (20.0%)
groups.
[0061] The secondary parameters monitored were used to support the
primary efficacy parameters (i.e., gross and microscopic (IHC)
lesions) responses due to vaccine and challenge for each treatment
group. Clinical observations made during the challenge period
indicated that the challenge controls had an overall higher score
when compared to the vaccine treatment groups 1-4, showing that the
challenge controls responded to the challenge. The supernatant
group did not seem to be as clinically affected. However, all
groups did have some pigs that did have some respiratory issues
with a cough, body condition, or behavior.
[0062] The treated groups saw an increase in average rectal
temperatures, above 104.degree. F., four hours after the first
vaccination event. Temperatures for the APP and omp groups spiked
at or above 104.9.degree. F., which was the cut-off of pyrexia
following four hours post vaccination, and lasted about twenty-four
hours for the omp group. Temperatures in the groups were returning
back to normal by day 1 or 3. For the booster event, temperatures
were only taken on day 28 and not at 4 hrs or on days 1, 3, and 5
post-vaccination as with the first vaccination. During the booster
period, on day 28 the APP group was statistically different
(p.ltoreq.0.05) when compared to the whole cell and supernatant
groups. During the challenge period, the challenge controls had an
overall higher rectal temperature at both the beginning and end of
the period. The whole cell group had the highest rectal
temperatures during days 38 and 39. The challenge control group had
the highest overall percent animals febrile (10/15) followed by the
whole cell (8/15), OMP (5/15), supernatant (2/15), and APP (1/15)
groups, respectively.
[0063] Bacterial isolation was highest in the lung and tonsil
samples. The highest recovery of A. suis occurred in the challenge
control group. The strict control group was negative for A. suis
except in nasal and tonsil samples, which had 3 out of 10 and 1 out
of 10 respectively. These results were recorded as a positive and
not verified by BCA or other means. Possible reasons for these
results could have been an inadvertent misread of the agar plate or
a cross-contamination event during the processing of the swabs.
Furthermore, three parameters had very low recovery rates of A.
suis which were the spleen, meninges, and heart blood, and thus,
were not included in the analysis. Overall the rate of septicemia
was low due to low recovery of A. suis in the visceral organs and
heart blood. There was also evidence that this challenge crossed
the blood brain barrier in three of the animals that were severely
infected. Other detected bacteria were Bordetella, alpha and beta
streptococci.
[0064] Serology testing using a western blot procedure was
performed to test for reactivity against the test article that was
used to vaccinate the treatment groups. The western blots showed
detectible seroconversion to the whole cell, supernatant, and omp
fractions at 1:100 dilution. The APP fraction had the best
reactivity and was detectable at a dilution of 1:1000, confirming
that the host recognized some part of the fractions used for
vaccination of each treatment group.
[0065] Overall the best protection occurred in the pigs vaccinated
with the APP and supernatant prototype vaccines. Comparatively,
neither the whole cell nor omp vaccines or LPS derived antigens
provided much protection from A. suis challenge. Details of the
challenge study are provided in the Examples below.
[0066] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs at the time of
filing. If specifically defined, then the definition provided
herein takes precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities, and plural terms shall include the singular.
Herein, the use of "or" means "and/or" unless stated otherwise. All
patents and publications referred to herein are incorporated by
reference.
EXAMPLES
[0067] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
A. Prototype Vaccines and Challenge Treatment
A. Preparation of Vaccine Prototypes
[0068] Growth of A. suis (KSU-1) Cultures:
[0069] One milliliter of A. suis (103007-1KSU) lot #201-9,10,11 was
used to inoculate 150 ml of BHI media (lot #195-121) in a 250 ml
spinner. The inoculated media was placed at 37.degree. C. at about
72 rpm for six hours. Afterwards, about 2.5 ml of the inoculated
media was added to about 2500 ml fresh BHI media. This step was
repeated until four batches of 2500 ml were prepared. The four
batches were incubated at 37.degree. C. overnight at about 72 rpm.
Two batches were used to isolate whole cell OMP, and supernatant
fractions, one batch was used to prepare a concentrated whole cell
fraction, and the final batch was used for LPS extraction.
[0070] Harvesting of Cell Fractions:
[0071] Optical densities of batches were read fifteen hours after
inoculation. Optical densities ranged from 0.775 to 0.815 at 600
nm. BAPs were streaked from each culture to verify that the
cultures were pure for A. suis.
[0072] To collect the OMP and supernatant fractions, two culture
batches were transferred to 500 ml centrifuge tubes, placed in a
JA-10 rotor, and spun at 10K for 10 minutes. The supernatants were
decanted, and the pellets were transferred into sterile 250 mL
Corning centrifuge tubes. The pellets were centrifuged at about
5000 rpm in a JA-10 rotor for 10 minutes, and the remaining
supernatants were removed. The pellets were pooled and placed at
-80.degree. C. until sarcosyl extraction (see below). The decanted
supernatants were pooled, and filtered through 0.45 .mu.m filters
(Nalgene 0.45 SFCA) into sterile 2 L Pyrex bottles. The filtered
supernatant was aliquoted into 2.times.850 cm RBs, 81.times.5 mL
vials, and 4.times.250 mL Nalgene bottles. Filtered supernatant was
stored at -20.degree. C. until use.
[0073] To collect the concentrated (10.times.) whole cell pellet
fraction, one culture batch was harvested as above, but the pellets
were resuspended in 300 mL TSP buffer. The solution was mixed well
and aliquoted into 50.times.2 mL vials and 4.times.50 mL vaccine
bottles. Resuspended whole cell pellets were stored at -80.degree.
C. until use.
[0074] To prepare the LPS fraction, A. suis culture was harvested
as described above. The pellets were resuspended in 1.times.PBS
buffer and re-centrifuged at 10K rpm for ten minutes. Supernatants
were decanted, and the pellets were resuspended in 1 L of
1.times.PBS buffer. An optical density reading at about 600 nm was
1.003, which is within the expected range of 0.8-1.200 for LPS
extraction. The LPS fraction was aliquoted into 3.times.250 mL
Nalgene bottles and stored at -80.degree. C. until use. The
remaining volume of LPS fraction was stored at 4.degree. C. for LPS
extraction.
[0075] KSU-1 Whole Cell Formalin-Inactivated Vaccine:
[0076] The optical density (OD) of the cell culture measured at 600
nanometer wavelength was at 0.775 at the time of harvest. In
previous studies, this optical density has correlated to
approximately 8.7 logs/mL based on the logarithmic estimate curve
to determine logs/mL based on colony forming units (cfu). The whole
cell fraction was concentrated 10.times. and frozen. The frozen
stock was then formalin-inactivated, and diluted (54 mLs of culture
into 26 mLs of 1.times. phosphate buffered saline (pbs), to 67.5%
of the total fraction. The inactivated, diluted vaccine stock was
then stored at 4.degree. C.
[0077] The whole cell formalin-inactivated vaccine stock was
removed from 4.degree. C. for formulation. Working in a bio-safety
hood 80 mLs of the material was aliquoted out into a sterile 100 mL
Pyrex bottle containing a magnetic stir bar. The bottle was placed
on a stir plate, and with continuous stifling, 20 mLs (20% v/v) of
the adjuvant (Emulsigen.RTM.-D) was added over 1 minute. The
adjuvanted prototype was then stirred for an additional 10 minutes.
The material was transferred to a 100 mL vaccine bottle where it
was capped and placed at 4.degree. C. Lot nos. for the whole cell
formalin-inactivated vaccine prototype were N201-110-WC-073108 (day
7) and N201-122-WC-082108 (day 21).
[0078] Sterility was verified by no growth on 5% Sheep Blood Agar
Plate (BAP) after 28 hrs and 30 hrs of 37.degree. C. aerobic
incubation for day 0 and day 21 respectively. Vaccine prototype
material was kept on ice prior to administration to the test
animals and during the entire vaccine administration procedure.
[0079] KSU-1 Supernatant Formalin-Inactivated Vaccine:
[0080] The supernatant prototype was formulated from the 10.times.
concentration step of the whole cell fraction preparation (see
above) by collecting the supernatant and filtering it through a
0.45 micro filter. The resulting filtered supernatant was frozen.
This supernatant was then formalin inactivated and stored at
4.degree. C. until formulated. The formulation step was as for the
whole cell formalin-inactivated vaccine. Lot nos. for the
supernatant formalin-inactivated vaccine prototype were
N201-110-Supe-073108 (day 7) and N201-122-Supe-082108 (day 21).
[0081] Sterility was verified, and the vaccine prototype material
was maintained prior to administration to the test animals and
during the entire vaccine administration procedure as previously
described.
[0082] KSU-1 OMP Fraction Vaccine:
[0083] The OMP fraction was produced from pellets prepared from a
KSU-1 culture (isolate BI-103007-1KSU) and stored at -80.degree. C.
by using the following OMP sarcosyl extraction procedure. Pellets
were thawed on the benchtop. The pellets were resuspended (22.5 g
wet weight) with 130 mL of 10 mM HEPES balanced salt solution and
aliquoted into four 50 mL conical Falcon tubes (40 mL/tube). (A 1
mL resuspended pellet sample was retained for protein
quantification and analysis.) The resuspended pellets were placed
in an ice bath and sonicated for 1 minute using (Large/Microtip)
probe (50 mL aliquots) with a sonication pulse setting of 1
pulse/sec. The sonication was repeated three times. (A 1 mL
post-sonication sample was retained.) The sonicated pellets were
centrifuged at 17,000.times.g for 20 min in 50 mL tubes.
Afterwards, the supernatants were decanted and pooled into a
separate container. Pellets were stored at 4.degree. C. until after
a bicinchoninic acid assay (BCA) and then discarded.
[0084] The pooled supernatants were run in an ultra centrifuge,
.about.0.0128 kg/tube, at a setting of 124,000.times.g (30,000
rpm), 1:10 hours, 4.degree. C., with maximum acceleration, and no
brake. Supernatants were decanted, pooled with the earlier
supernatant, and stored at 4.degree. C. About 1 mL of 10 mM HEPES
was added on top of each pellet in the ultra centrifuge tubes, and
the pellets were incubated overnight at 4.degree. C. to loosen the
pellets from the tubes. Another 2 mL of 10 mM HEPES was added to
each tube to resuspend the pellets. Each resuspended pellet
solution was q.s. to 6 mL (total vol.) with 10 mM HEPES, and 6 mL
of 2% Sarcosyl was added to each. Solutions were incubated for 30
min at room temperature. Afterwards, the tubes were balanced in
ultra centrifuge buckets (.about.0.0128 kg/tube) using 2% sarcosyl
and run in an ultra centrifuge at 124,000.times.g, 1:10 hour,
4.degree. C., with maximum acceleration, and no brake. The
supernatants were collected into a vessel, and 1 mL of 10 mM HEPES
was added to each pellet. The OMP pellets were stored are 4.degree.
C. for 1 hour prior to measuring protein concentration or
visualization of proteins.
[0085] Duplicate samples (1:10 dilution) were run on either a
reduced MOPS 10-12% Bis-Tris SDS PAGE gel or a NuPAGE 4-12%
Bis-Tris gel to visualize protein bands. Total protein
concentrations of the final OMP stock were determined using the BCA
and were determined prior to formulation with adjuvant. Based on
the values obtained by BCA, the total mass of protein contained in
the OMP extraction stock was 1.75 .mu.g/mL. Based on this value,
the potency of the formulated vaccine OMP prototype was 250
.mu.g/dose. The resuspended, extracted OMP fraction was aliquoted
into 200 .mu.l samples, labeled, and frozen at -70.degree. C.
[0086] Five vials containing 0.5 mLs of the extracted OMP fraction
were removed from -70.degree. C. These vials were thawed at room
temperature. Working in a bio-safety hood 23.25 mLs of 1.times.
phosphate buffered saline (pbs) and 2.35 mLs of the OMP material
were aliquoted into a sterile 100 mL Pyrex bottle containing a
magnetic stir bar. The bottle was placed on a stir plate, and with
continuous stirring, 6.4 mLs (20% v/v) of the adjuvant
(Emulsigen.RTM.-D) were added over 1 minute. The adjuvanted OMP
prototype vaccine was then stirred for an additional 10 minutes.
The OMP prototype vaccine was transferred to a 60 mL vaccine bottle
where it was capped and placed at 4.degree. C. Lot nos. for the OMP
vaccine prototype were N201-110-OMP-073108 (day 7) and
N201-122-OMP-082108 (day 21).
[0087] Sterility was verified, and the vaccine prototype material
was maintained prior to administration to the test animals and
during the entire vaccine administration procedure as previously
described.
[0088] INGELVAC.RTM. APP-ALC Vaccine:
[0089] One bottle of APP-ALC was removed from -70.degree. C. and
thawed in luke warm water. Once thawed, a sample was removed using
a 16 gauge sterile needle and placed in a vaccine bottle for
potency testing. The vaccine bottle was placed at 4.degree. C.
Titrations of INGELVAC.RTM.APP-ALC were performed on Days 0 and 21
for determining colony forming units (CFU) per dose. The samples
were serially diluted out 10-fold and plated onto Mueller Hinton
Chocolate Agar plates. Three reps were performed and allowed to
incubate at 37.degree. C. for 24 hrs before determining the CFU.
Colony forming units were respectively at Day 0: 9.99 logs/dose and
at Day 21: 10.2 logs/dose. Lot nos. for the INGELVAC.RTM. APP-ALC
vaccine were N201-110-APP-073108 (day 0) and N201-122-APP-082108
(day 21).
[0090] Vaccine prototype material was maintained prior to
administration to the test animals and during the entire vaccine
administration procedure as previously described.
[0091] Placebo Vaccine
[0092] Working in a bio-safety hood, 60 mLs of 1.times. phosphate
buffered saline (pbs) was aliquoted out into a sterile 100 mL Pyrex
bottle containing a magnetic stir bar. The bottle was placed on a
stir plate, and with continuous stirring, 15 mLs (20% v/v) of the
adjuvant (Emulsigen.RTM.-D) was added over 1 minute. The adjuvanted
prototype was then stirred for an additional 10 minutes. The
material was transferred to 100 mL vaccine bottle where it was
capped and placed at 4.degree. C. The Lot no. for the placebo
vaccine was N 201-109.
[0093] Sterility was verified, and the placebo prototype material
was maintained prior to administration to the test animals and
during the entire vaccine administration procedure as previously
described.
B. Preparation of Challenge Treatment
[0094] The challenge strain used was A. suis Isolate ISU-8594 p5.
The challenge material was produced by inoculating a 1 liter Belco
spinner flask containing 800 mLs of Brain Heart Infusion Porcine
(BHI-Porcine) media with 5 mLs of ISU-8594 p4. The spinner was
placed into a 37.degree. C. incubator at .about.100 rpm. The
culture was grown to an optical density (OD600) of 0.109-about 5
hrs and 30 minutes post inoculation. The culture was then
transferred to 6.times.100 mL vaccine bottles containing 100 mLs
total volume that were stoppered and capped. Five of the bottles
were placed into a cooler containing ice packs and one bottle which
served to represent the other five bottles for titrations was
placed in another cooler containing ice packs. The titer of the
challenge material was determined by CFU count generated from
plating serial 10-fold dilutions onto 5% sheep blood agar plates.
The challenge titer obtained was 1.69.times.10.sup.8 cfu(s)/mL or
9.0 logs/6 mL dose. The Lot no. for the challenge material was
N201-138.
[0095] Challenge material was kept on ice prior to administration
to the test animals and during the entire challenge procedure.
Example 2
Vaccination of Pigs
[0096] A mixture of female and neutered male porcine animals of a
commercial cross and 3 weeks.+-.7 days of age were obtained from
Wilson Prairie View Farms. Animals were healthy with no evidence of
clinical respiratory disease and negative for bacterial respiratory
pathogens such as A. pleuropneumonia, H. parasuis, S. suis, P.
multocida, B. bronchiseptica, E. rhusiopathiae and A. suis. While
allowance was made for animals to be excluded from the study if and
when health problems unrelated to vaccination or challenge became
apparent, no animals were removed.
[0097] At the time of arrival to the test site, all animals
received a 1 mL shot of EXCENEL.RTM. (a short acting antibiotic).
The animals were housed at the test site until the study was
terminated. The animals were ear-tagged upon arrival, and housed
appropriately for species, age, size, and condition. Challenge and
strict control pigs were housed together in separate pens in a
separate building away from the other treatment groups. At the time
of challenge, the challenge controls were moved into the same
building as the treated groups. All treatment groups were housed in
separate pens throughout the study. The animals were provided with
a ration that was free of antibiotics and appropriate for species,
age, size, and condition of the animals. The animals were in good
health and nutritional status at initiation of the study. A health
examination was conducted by a clinical veterinarian according to
generally accepted veterinary practice on each animal prior to
inclusion in the study.
[0098] A computer random number generator (Microsoft Office Excel)
was used to assign each animal a unique random number. The random
number was then sorted into ascending order. Assignments occurred
by allocating the experimental units across treatment groups
starting with the lowest block of random numbers and assigning them
to treatment groups 1 through 6. Increasing in random number, the
next block of experimental units were then assigned to treatment
groups and so on until all animals were assigned to a treatment
group.
[0099] This efficacy study consisted of 6 treatment groups of
weaned 3 week-old.+-.7 days of age pigs. Treatment group 1 (15
pigs) received a 1.times.2 mL intramuscular (IM) dose of
formalin-inactivated whole cell A. suis on days 0 and 21 of the
study, respectively. Treatment group 2 (15 pigs) received a
1.times.2 mL IM dose of formalin-inactivated A. suis culture
supernatant on days 0 and 21 of the study, respectively. Treatment
group 3 (15 pigs) received a 1.times.2 mL IM dose of A. suis outer
membrane protein (omp) on days 0 and 21 respectively. Treatment
group 4 (15 pigs) received a 1.times.2 mL IM dose of INGELVAC.RTM.
APP-ALC on days 0 and 21 respectively. Treatment group 5 (15 pigs)
designated as "challenge controls" received a 1.times.2 mL dose of
placebo by IM route of administration on days 0 and 21
respectively. Treatment group 6 (10 pigs) were designated "strict
controls" and did not receive vaccine or placebo treatment. All
groups were observed for 41 days.
[0100] On Day 35 of the study, Groups 1-5 received a 6 mL dose/pig
(3 mL applied to each nostril per pig) of A. suis strain ISU-8594
via intranasal (IN) inoculation containing 1.times.10.sup.9.0
logs/dose. Group 6, the "strict control" group, did not receive any
treatment or challenge.
[0101] Rectal temperatures were collected from all animals prior to
treatment on Day 0; 4 hours post inoculation; and on 1 day(s) post
inoculation (DPI), 3, 5, 7, 14, 21, 28, 34, 35, 36, 37, 38, 39, 40
and 41 DPI.
[0102] Venous whole blood (6-10 mL) was collected from each animal
prior to treatment on Day 0 and weekly (Days 7, 14, 21, 28, 34 and
41) for future serological testing.
[0103] Injection sites were examined prior to treatment and at 4,
24, 48, and 72 hours post inoculation in all pigs of Groups 1-5.
The injection sites were examined for swelling, hardness, and size.
The data were documented. The following scoring system was used for
injection site observations: swelling (0=none, 1=swelling present),
appearance (0=normal, 1=hard, 2=soft, 3=abscessed, 4=draining),
size (0=normal, for all others, the length by width dimensions in
centimeters (i.e. length.times.width.times.diameter) were
recorded).
[0104] Clinical observations were performed daily from Day 0 to Day
41 for any signs of respiratory distress including labored
breathing, sneezing, coughing, altered respiratory movements,
anorexia, lameness, swelling of joints, dehydration, ability to
stand, paddling, moribund for 2 or more consecutive days, or
death.
[0105] Animals that had severe clinical symptoms during the study
were humanely euthanized and necropsied to determine the cause of
death. On Day 41 of the study, all animals were euthanized and all
sections of the lung were scored for determining the percentage of
lung pathology. Fresh and fixed (lungs only) samples of the lung (3
lobes with lesions, if present), liver, kidney, spleen, tonsil, and
swabs of nasal turbinates, trachea, bronchi, meninges, and heart
blood were collected from each animal and cultured for bacterial
isolation and histopathology (lungs only).
Example 3
Evaluating Efficacy of Prototype Vaccines
[0106] A. Statistical Analysis
[0107] Statistical Analysis was performed using SAS version 9.1.3
for data management and analysis. Summary statistics including
mean, standard deviation, standard error, median, range, 95%
confidence intervals, coefficient of variation, and frequency
distributions were generated for all data where appropriate.
Analyses included all pairwise comparisons between all piglet
treatment groups. All tests for significance were two-tailed with a
p.ltoreq.0.05 level to determine differences between treatment
groups.
[0108] Primary Efficacy Parameters were non-normally distributed
microscopic lesion scores and gross lesion scores that were
compared by Wilcoxon Two-Sample Test and Fisher's Exact Test.
Secondary Efficacy Parameters were (1) clinical signs that were
compared using the Wilcoxon Two-Sample Test; (2) pyrexia that was
compared using the Fisher's Exact Test and ANOVA; and (3) bacterial
isolation was compared using the Fisher's Exact Test.
B. Moribund Animals
[0109] Two animals were removed from the study after study
initiation. Animal ID #31 (treatment group 4) was found dead on day
11 of the study. Gross examination showed that this pig was in poor
condition and thin. Its lungs had red and purple discoloration, and
its abdomen had purulent exudates with fibrous peritonitis. The
presumptive diagnosis was HPS or a streptococcal infection.
Bacteriology cultures were performed on the fresh tissue and swab
samples using standard techniques accepted in the field. The
recovered bacterial colonies were analyzed, and the analysis found
that the pig was infected with Arcanobacterium pyrogenes, Aeromonas
hydrophilia, and Citrobacter freundii. Histopathology on fixed lung
samples indicated no evidence of pneumonia, necrosis, pleuritis, or
bacterial colonies present.
[0110] Animal ID #32 (treatment group 1) was found dead on day 36
one day post-challenge. Gross examination showed that this pig's
lungs had severe fibrinouses and fluid in the chest cavity. Its
lungs had consolidation and large hemorrhagic areas. The
presumptive diagnosis was acute death due to A. suis challenge.
Bacteriology cultures were performed on the fresh tissue and swab
samples. Actinobacillus suis was recovered in all tissue and swab
samples except the spleen and trachea. Histopathology on fixed lung
samples indicated evidence of severe pneumonia, necrosis,
pleuritis, and bacterial colonies present.
C. General Observations
[0111] Animals were observed daily from vaccination/placebo
administration to challenge (days 0 through 35) for adverse events
attributed to treatment with test/control articles or other
non-treatment derived health abnormalities.
[0112] General health observations for pigs in challenge control
and strict control groups (groups 5 and 6) were as follows: Pig #71
(challenge control) started showing signs of lameness on its right
front on day 7 and persisted to day 24 before symptoms ceased. The
rest of the pigs were normal for this period of observation.
[0113] General health observations for pigs in vaccine treatment
groups (groups 1-4) were as follows: Pig ID #51 (whole cell, group
1) showed poor body condition on day 1 and persisted for three
days. In treatment group 4 (APP, group 4) Pig ID #31 was removed
from the study as stated above. The remaining pigs in groups 1
through 4 had normal health observations.
[0114] Injection site reactions for IM-vaccinated treatment groups
1, 2, 3 and 4 were as follows: Pigs #35 (supernatant, group 2) and
#10 (APP, group 4) had swelling of a 1.times.1 cm.sup.2 area
recorded on days 1, 2, and 3 of the study. No injection site
evaluations were recorded for the day 21 vaccination event.
Clinical observations revealed swelling in the neck in pig #10
(APP, group 4) on days 4 and 5, pig #20 (APP, group 4) on days 26
and 29, pig #40 (APP, group 4) on days 19 through 35, pig #41 (APP,
group 4) on days 26 through 35, and pig #46 (APP, group 4) on days
14 through 35. All remaining animals had a normal, healthy
disposition during this observation period.
[0115] At time of necropsy, pigs #69 and #75 both in treatment
group 1 (whole cell) were recorded as having neck abscesses at the
site of injections. No other adverse injection site recordings were
reported during necropsy.
D. Primary Efficacy Parameters
[0116] 1. Gross Lesions
[0117] At necropsy (day 41 of the study), the lungs were removed
from each pig and examined for gross lesions. Individual lobes were
scored for percent involvement, and a total score was assigned to
each individual pig. Table 1 shows the average lung lesion scores
for each treatment group and the number of animals with a positive
score per group.
TABLE-US-00001 TABLE 1 Average gross lung lesion scores by
treatment group and number of animals with a positive gross score
within groups. Percent with Group Treatment N Mean Lesion Present 1
Whole Cell 15 26.73 66.67% (10/15) 2 Supernatant 15 12.68 60.00%
(9/15) 3 OMP 15 21.69 80.00% (12/15) 4 APP 14 16.25 50.00% (7/14) 5
Challenge 15 28.67.sup.a 86.67.sup.a (13/15) 6 Strict 10 0.00.sup.a
0.00.sup.a (0/10) .sup.aGroups 5 vs. 6 comparisons are
statistically significant different (p < 0.05, Wilcoxon Two
Sample Test and Fishers Exact Test where appropriate).
[0118] Using the Wilcoxon Two-Sample Test and Fishers Exact Test on
the number of positive lesions per group, statistical analyses were
run to compare the means among treatment groups 5 vs. 6 and groups
1-4 vs. 5 on average gross lesion scores. Treatment group 6 (strict
controls) was negative for gross lesion development (0) and the
number of percent positive scores (0%). Evaluation of groups 1-5
for average lung lesion scores and percent with a lesion ranged
from 12.68 to 28.67 out of a possible 100 and from 50.00% to 86.67%
respectively.
[0119] Treatment group 5 (challenge), received the highest average
gross lung lesion score (28.67) and percent with a lung lesion
score (86.67%) compared to the other treatment groups. Treatment
group 5 was significantly different (p.ltoreq.0.05) from treatment
group 6, when comparing average gross lung lesion scores and
percent positive with a lesion. Treatment group 2 (supernatant)
received the lowest lesion score (12.68), but had a percent lesion
score that was the second lowest (60.00%) compared to treatment
group 4 (APP) which had the lowest percent lesion score (50.00%).
Comparisons of groups 4 vs. 5 for percent with a lung lesion score
was almost statistically different (p=0.0502). Furthermore,
treatment group comparisons for gross lesion score of groups 4 vs.
5 and groups 2 vs. 5 were not statistically different,
(p.ltoreq.0.0840) and (p.ltoreq.0.0685) respectively. Numerically,
treatment group 1 (whole cell) had the second highest average lung
lesion score of 26.73 followed by the treatment group 3 (omp) with
a score of 21.69. Treatment group 3 (omp) had the second highest
number of percent lesions present with 80.00% followed by treatment
group 1 (whole cell) with 66.67%.
[0120] 2. Microscopic Lesions
[0121] Lung sections were collected, fixed, and submitted to the
Iowa State Veterinary Diagnostic Laboratory for evaluation of
non-specific microscopic lesion development (i.e. pneumonia,
necrosis, and pleuritis) and presence of bacterial colonies. Scores
were based on a nominal scale of 1 to 3 with 1 being mild, 2 being
moderate, and 3 being severe. Groups were compared based on average
lesion severity and presence of bacterial colonies (bugs) present
within each tissue, and the frequency of positives for each group
are shown in Table 2.
TABLE-US-00002 TABLE 2 Average microscopic lesion scores by
treatment group and number of animals with a positive microscopic
score within groups. Broncho Pneumonia Broncho Bacterial Grp
Treatment N lesion Necrosis Pleuritis Pneumonia Necrosis Pleuritis
Colonies 1 Whole 15 1.07 0.67 1.67 53.33% 40.00% 60.00% 20.00% Cell
(8/15) (6/15) (9/15) (3/15) 2 Supernatant 15 0.87 0.8 1.13 46.67%
40.00% 46.67% 33.33% (7/15) (6/15) (7/15) (5/15) 3 OMP 15 1.60
1.47.sup.c 1.60.sup.c 66.67% 60.00% 66.67% 46.67% (10/15) (9/15)
(10/15) (7/15) 4 APP 15 0.93 0.47.sup.c 0.67.sup.b,c 46.67% 26.67%
46.67% 20.00% (7/15) (4/15) (7/15) (3/15) 5 Challenge 15 1.27.sup.a
0.93.sup.a 1.93.sup.a,b 66.67%.sup.a 46.67%.sup.a 66.67%.sup.a
40.00% (10/15) (7/15) (10/15) (6/15) 6 Strict 10 0.00.sup.a
0.00.sup.a 0.00.sup.a 0.00%.sup.a 0.00%.sup.a 0.00%.sup.a 0.00%
Control (0/10) (0/10) (0/10) (0/10) .sup.aGroups 5 vs. 6
comparisons are statistically significant different (p < 0.05,
Wilcoxon Two Sample Test and Fishers Exact Test where appropriate).
.sup.bGroups 4 vs. 5 comparisons are statistically significant
different (p < 0.05, Wilcoxon Two Sample Test and Fishers Exact
Test where appropriate). .sup.cGroups 3 vs. 4 comparisons are
statistically significant different (p < 0.05, Wilcoxon Two
Sample Test and Fishers Exact Test where appropriate).
[0122] Using the Wilcoxon Two-Sample Test, statistical analyses
were run to compare the means among treatment groups on average
microscopic lung lesions scores. The Fisher Exact Test was also
performed on the number of positive scores for the percent positive
animals per group for each parameter. Statistical differences
(p.ltoreq.0.05) were found when comparing treatment groups 5
(challenge) vs. 6 (strict control) for bronchopneumonia, necrosis,
and pleuritis lesion scores, and percent positive. The scores were
zero in all parameters for treatment group 6 (strict controls)
(Table 2).
[0123] The highest bronchopneumonia lesion scores (1.60) were found
in treatment group 3 (omp) followed by (1.27) treatment group 5
(challenge). The lowest received score (0.87) was in group 2
(supernatant) followed by (0.93) group 4 (APP). Scores for
bronchopneumonia ranged from 0.87 to 1.60. Comparing necrosis
lesion scores for treatment groups 1-5 ranged from 0.47 found in
group 4 (APP) to 1.47 found in group 3 (omp). Statistical
differences (p.ltoreq.0.05) were found when comparing treatment
groups 3 vs. 4 for necrosis. Further comparisons of treatment
groups 1-5 for the pleuritis parameter revealed lesion scores that
ranged from 0.67 in group 4 (App) to 1.93 in group 5 (challenge).
Statistical differences (p.ltoreq.0.05) were found when comparing
treatment groups 3 vs. 4 and 4 vs. 5 for pleuritis. Comparison of
treatment groups 1 vs. 4 was very close to being statistically
different (p=0.0544).
[0124] Numerical comparisons of percent positives showed that when
compared to treatment groups 1, 2, 4, and 5 treatment group 3 (omp)
received the highest scores for bronchopneumonia, necrosis,
pleuritis, and bacterial colonies of 66.67%, 60.00%, 66.67%, and
46.67% respectively. The highest scores were shared with treatment
group 5 (challenge) for bronchopneumonia and pleuritis parameters.
Furthermore, comparisons of percent positives showed that when
compared to the treatment groups 1, 2, 3, and 5 the lowest scores
were in treatment group 4 (APP) for bronchopneumonia, necrosis,
pleuritis, and bacterial colonies of 46.67%, 26.67%, 46.67%, and
20.00% respectively. Treatment group 2 (supernatant) had the same
lowest scores in bronchopneumonia and pleuritis parameters as group
4, and group 2 and group 1 (whole cell) had the same frequency of
bacterial colonies present. No statistical differences were found
for percent positives in the parameters listed in Table 2.
E. Secondary Efficacy Parameters
[0125] Secondary parameters were used to support primary efficacy
parameters in this study. Statistical analyses were performed on
the following secondary parameters.
[0126] 1. Clinical Observations
[0127] Observations for clinical signs were made from the day of
vaccination throughout the study (days 0 through 41). Four main
parameters were scored: body condition (gauntness) on a scale of 1
to 3 with 1 being normal and 3 being dead; behavior (depression);
locomotion; and respiration, each on a scale of 1 to 4 with 1 being
normal and 4 being dead. An average clinical score was used from
all 4 parameters by which a normal, healthy animal received a score
of 1 while a severely affected animal (dead) could have a maximum
score of 3.75. Table 3 shows the average scores by treatment group.
Using the Wilcoxon Two-Sample Test, statistical analysis was only
performed to compare groups 1 through 5 on average clinical
observation scores.
TABLE-US-00003 TABLE 3 Daily average clinical scores for
pre-challenge and post-challenge periods within each treatment
group. Pre- Post Challenge Post Challenge Challenge Days Day Day
Day Day Day Day Days Grp Treatment (0-35) 36 37 38 39 40 41 (36-41)
1 Whole Cell 1.001.sup.a,b 1.25.sup.b 1.04 1.00 1.02 1.04 1.07
1.068.sup.a 2 Supernatant 1.000.sup.a,b 1.03 1.03 1.00 1.00.sup.a
1.00.sup.a 1.03.sup.a 1.017.sup.a 3 OMP 1.000.sup.a,b 1.07.sup.b
1.03 1.05 1.00.sup.a 1.05 1.13 1.058.sup.a 4 APP 1.011.sup.b
1.00.sup.a,b 1.02 1.00 1.02 1.04 1.14 1.036.sup.a 5 Challenge
1.013.sup.a 1.12.sup.a 1.08 1.12 1.28.sup.a 1.14.sup.a 1.14.sup.a
1.148.sup.a *6 Strict 1.000 1.00 1.00 1.00 1.00 1.00 1.00 1.000
*Treatment group 6 was not included in the statistical analysis.
.sup.aGroups 1-4 vs. 5 comparisons are statistically significant
different (p < 0.05, Wilcoxon Two Sample Test where
appropriate). .sup.bGroups 1-3 vs. 4 comparisons are statistically
significant different (p < 0.05, Wilcoxon Two Sample Test where
appropriate).
[0128] Comparison of the treatment groups 1-5 during the
pre-challenge period revealed that during this time period compared
to the treated groups 1-4 the untreated challenge controls had the
highest clinical scores of 1.013. Treatment group 4 (APP) had the
second highest mean clinical score of 1.011 followed by groups 1, 2
and 3 with scores of 1.001, 1.000, and 1.000 respectively.
Statistical differences (p.ltoreq.0.05) were found when comparing
treatment groups 1-3 vs. group 5 from an overall mean score for the
pre-challenge period. In addition statistical differences
(p.ltoreq.0.05) were found when comparing groups 1-3 vs. group 4.
No statistical differences were found when comparing group 4 vs.
group 5.
[0129] During the challenge period it was noted that treatment
group 5 (challenge) had the highest overall score of 1.148 followed
by groups 1, 3, 4, and 2 with scores of 1.068, 1.058, 1.036, and
1.017 respectively. For overall average clinical scores,
statistical differences (p.ltoreq.0.05) were found when comparing
groups 1-4 vs. group 5. During the challenge period, treatment
group 5 (challenge) had the highest scores on days 37 through 41.
Treatment group 1 (whole cell) had the highest score on day 36.
Furthermore, treatment group 1 (whole cell) scores fell numerically
following day 36 spike from 1.25 to a range of 1.00 to 1.07. The
lowest overall score was in treatment group 2 (supernatant), and it
was statistical different (p.ltoreq.0.05) compared to group 5
(challenge) on days 39, 40, and 41. Further comparison of group 4
(APP) showed that on day 36, one day post challenge, there was a
statistical difference (p.ltoreq.0.05) compared to group 5
(challenge). Also, on day 36 groups 1 and 3 were statistical
different (p.ltoreq.0.05) compared to group 4 (APP).
[0130] 2. Rectal Temperatures
[0131] To monitor the effects of vaccination and challenge, rectal
temperatures were taken at different time points throughout the
study (Days 0, 0+4 hrs, 1, 3, 5, 7, 14, 21, 28, 34, 35, 36, 37, 38,
39, 40, and 41). Temperature spikes greater than (104.9.degree. F.)
were considered to be significant. The number of animals with
temperatures exceeding the cut-off are listed in Table 4. Using
Fisher's Exact Test and ANOVA, statistical analyses were only
performed to compare pyrexia scores for groups 1 through 5.
[0132] Referring to FIG. 1 and Table 4 for the first vaccination
time period of day 0 to 21, there was a general spike in rectal
temperature in all treatment groups with a larger spike
.gtoreq.104.9.degree. F. at time 4 hours post vaccination in
treatment groups 3 (omp) and 4 (APP) and continued to day 1 for
group 3 (omp). In group 1 (whole cell) there was a decrease in
temperature on day 7 to 102.degree. F., and statistical differences
were observed (p.ltoreq.0.05) when comparing group 1 vs. groups 2-5
on this day. Furthermore, the treated animal temperatures in groups
1-4 were numerically less than the untreated groups 4-5 on day 7.
On day 21 of the study the treated animals had numerically lower
temperatures than the untreated and were found to be statistically
different (p.ltoreq.0.05) when comparing groups 1-4 vs. group
5.
TABLE-US-00004 TABLE 4 Rectal temperatures (.degree. F.) from day 0
to day 21 (first vaccination period). Day Day Day Grp Treatment Day
0 0 + 4 hrs. Day 1 Day 3 Day 5 Day 7 14 21 1 Whole Cell 103.1
104.1.sup.b 103.5.sup.b 103.3 103.5 102.0.sup.a,b 103.4.sup.a,b
103.7.sup.a,b 2 Supernatant 103.2 104.2.sup.c 103.6.sup.c 103.3
103.3 102.8.sup.a,b 103.6 103.3.sup.a,b 3 OMP 103.2
104.9.sup.a,b,c,d 104.9.sup.a,b,c 103.3 103.3 102.8.sup.a,b 103.6
103.5.sup.a 4 APP 103.3 105.1.sup.a,b,d 103.9.sup.c 103.2 103.5
102.9.sup.a,b 103.8.sup.b 103.5.sup.a 5 Challenge 103.1 103.8.sup.a
103.5.sup.a 103.5 103.6 103.6.sup.a 103.7.sup.a 104.1.sup.a 6
Strict 103.1 103.8 103.2 103.5 103.7 103.5 104.1 104.1 * Treatment
group 6 was not included in the statistical analysis. .sup.aGroups
1-4 vs. 5 comparisons are statistically significant different (p
< 0.05, ANOVA Test where appropriate). .sup.bGroups 1 vs. 2-4
comparisons are statistically significant different (p < 0.05,
ANOVA where appropriate). .sup.cGroups 2 vs. 3-4 comparisons are
statistically significant different (p < 0.05, ANOVA where
appropriate). .sup.dGroups 3 vs. 4 comparisons are statistically
significant different (p < 0.05, ANOVA where appropriate).
[0133] Referring to FIG. 1 and Table 5, for the booster time period
of day 22 through day 35 it was observed that treatment group 4
(APP) had the highest numerical rectal temperature on day 28 of the
study. Statistical differences were observed (p.ltoreq.0.05) on day
28 when comparing treatment groups 1 and 2 vs. group 4.
TABLE-US-00005 TABLE 5 Rectal temperatures in degrees Fahrenheit
(.degree. F.) from day 22 to day 35 (booster period). Group
Treatment Day 28 Day 34 Day 35 1 Whole Cell 103.7.sup.a 103.5 103.9
2 Supernatant 103.6.sup.b 103.7 104.0 3 OMP 103.9 103.6 103.8 4 APP
104.3.sup.a,b 103.5 103.7 5 Challenge 103.9 103.5 103.7 6 Strict
103.9 103.5 103.7 * Treatment group 6 was not included in the
statistical analysis. .sup.aGroups 1 vs. 2-4 comparisons are
statistically significant different (p < 0.05, ANOVA where
appropriate). .sup.bGroups 2 vs. 3-4 comparisons are statistically
significant different (p < 0.05, ANOVA where appropriate).
[0134] Referring to FIG. 1 and Table 6, for the challenge time
period days 36 through day 41 it was observed that treatment group
5 (challenge) had the highest numerical rectal temperatures on days
36, 37, 40, and 41; whereas, treatment group 1 (whole cell) had the
highest rectal temperatures on days 38 and 39. Furthermore,
treatment group 4 (APP) had the lowest overall rectal temperatures
on days 36, 37, 38, and 41. In addition treatment group 5
(challenge) had the same lowest temperature on day 38 as treatment
group 4, and groups 2 and 3 had the lowest temperatures on days 39
and 40 respectively. Both groups 3 and 5 had an initial rise in
temperature one day post-challenge that then fell for group 3;
however, the temperature for group 5 rose again on day 40.
TABLE-US-00006 TABLE 6 Rectal temperatures (.degree. F.) from day
36 to day 41 (challenge period) and number of animals with pyrexia
(.gtoreq.104.90.degree. F.) in parentheses for at least one day
from day of challenge to necropsy. Day Day Day Day Day Day Group
Treatment 36 37 38 39 40 41 % Present 1 Whole Cell 104.0 104.1
103.9.sup.b 104.0 103.7 103.8.sup.b 53.33%.sup.b (8/15) 2
Supernatant 103.7.sup.a 103.8.sup.a 103.7 103.5 103.9 103.9.sup.c
13.33%.sup.a,b (2/15) 3 OMP 104.3 103.9.sup.a 103.8 103.8
103.4.sup.a 103.8.sup.c 33.33% (5/15) 4 APP 103.5.sup.a 103.6.sup.a
103.5.sup.b 103.7 103.6.sup.a 103.1.sup.a,b,c 7.14%.sup.a,b (1/14)
5 Challenge 104.6.sup.a 104.4.sup.a 103.5 103.7 104.1.sup.a
104.0.sup.a 66.67%.sup.a (10/15) 6 Strict 103.7 103.8 103.7 103.6
103.6 103.5 103.7 * Treatment group 6 and Spleen, Meninges, and
Heart Blood were not part of the statistical analysis. .sup.aGroups
1-4 vs. 5 comparisons are statistically significant different (p
< 0.05, ANOVA and Fishers Exact Test where appropriate).
.sup.bGroups 1 vs. 2-4 comparisons are statistically significant
different (p < 0.05, ANOVA and Fishers Exact Test where
appropriate). .sup.cGroups 2-3 vs. 4 comparisons are statistically
significant different (p < 0.05, ANOVA and Fishers Exact Test
where appropriate).
[0135] Further evaluation of the challenge period showed that the
challenge group 5 received the highest percent positive of animals
that were febrile with a value of 66.67%, which was statistically
different (p.ltoreq.0.05) when compared to groups 2 and 4. The
second highest score received was in treatment group 1 with a score
of 53.33%, which was also statistically different (p.ltoreq.0.05)
when compared to groups 2 and 4. Groups 4 (APP) and 2 (supernatant)
received the lowest scores of 7.14% and 13.33% respectively.
Treatment group 3 (omp) had a score of 33.33%.
[0136] 3. Bacterial Isolation
[0137] At necropsy, swabs of the nasal cavity, trachea, bronchi,
meninges, and heart blood were collected for bacterial isolation.
Sheep blood agar plates (5% with TSA) and MacConkey agar plates
were inoculated with each swab, streaked for isolation and
incubated overnight at 37.degree. C. Blood agar plates were placed
under anaerobic and aerobic conditions, and the MacConkey agar
plates were placed under aerobic conditions only. In addition,
chocolate agar plates were used only for culturing lung samples and
incubated at 37.degree. C. under aerobic conditions. Fresh tissue
samples of the lung (3 lobes from sections containing lesions),
liver, kidney, and tonsil were obtained. Swabs of each tissue were
used to inoculate agar plates for bacterial isolation. Plates were
incubated along with the swab samples mentioned above, and plates
were observed for presence of A. suis 24 hours post incubation.
Biochemical analysis was done on a random sample in each of the
treatment groups to confirm the presence of A. suis. Statistical
analyses were only performed to compare groups 1 through 5 on
bacterial isolation scores using the Fisher's Exact Test. The
spleen, meninges, and heart blood were not statistically analyzed.
Tables 7a and 7b provides a summary of the bacteriology
results.
TABLE-US-00007 TABLE 7a Number of percent positive A. suis animals
in treatment groups by bacterial isolation. Group Treatment Lung
Liver Kidney Tonsil Nasal 1 Whole 40% 6.67% 6.67% 66.67% 13.33%
Cell (6/15) (1/15) (1/15) (10/15) (2/15) 2 Super- 33.33% 0.00%
13.33% 80.00% 13.33% natant (5/15) (0/15) (2/15) (12/15) (2/15) 3
OMP 33.33% 0.00% 0.00% 60.00% 6.67%a (5/15) (0/15) (0/15) (9/15)
(1/15) 4 APP 33.33% 6.67% 6.67% 60.00% 0.00%a (5/15) (1/15) (1/15)
(9/15) (0/15) 5 Challenge 73.33% 13.33% 20.00% 86.67% 46.67a
(11/15) (2/15) (3/15) (13/15) (7/15) *6 Strict 0.00% 0.00% 0.00%
0.00% 20.00% (0/10) (0/10) (0/10) (0/10) (3/10) *Treatment group 6
and Parameters Spleen, Meninges, and Heart Blood were not part of
the statistical analysis. .sup.aGroups 1-4 vs. 5 comparisons are
statistically significant different (p < 0.05, Fishers Exact
Test where appropriate).
TABLE-US-00008 TABLE 7b Number of percent positive A. suis animals
in treatment groups by bacterial isolation. *Heart Group Treatment
Trachea Bronchi *Spleen *Meninges blood 1 Whole 6.67% 13.33% 0.00%
6.67% 6.67% Cell (1/15) (2/15) (0/15) (1/15) (1/15) 2 Super- 6.67%
0.00% 0.00% 0.00% 0.00% natant (1/15) (0/15) (0/15) (0/15) (0/15) 3
OMP 6.67% 13.33% 6.67% 0.00% 0.00% (1/15) (2/15) (1/15) (0/15)
(0/15) 4 APP 20.00% 20.00% 0.00% 6.67% 0.00% (3/15) (3/15) (0/15)
(1/15) (0/15) 5 Challenge 20.00% 26.67% 6.67% 6.67% 6.67% (3/15)
(4/15) (1/15) (1/15) (1/15) *6 Strict 6.67% 0.00% 0.00% 0.00% 0.00%
(1/10) (0/10) (0/10) (0/10) (0/10) *Treatment group 6 and
Parameters Spleen, Meninges, and Heart Blood were not part of the
statistical analysis. .sup.aGroups 1-4 vs. 5 comparisons are
statistically significant different (p < 0.05, Fishers Exact
Test where appropriate).
[0138] The recovery of A. suis in each of the target areas was the
highest in treatment group 5 (challenge) compared to the other
groups (1-4). The highest percentage of A. suis recovery was found
in the tonsil followed by the lung. The recovery of A. suis was low
in the spleen, meninges, and heart blood parameters with only a
maximum recovery yield of 6.67% among the treatment groups. The
strict controls, group 6, had three out of ten pigs positive for A.
suis in nasal tissue, and one pig positive for tracheal tissue.
Statistical differences (p.ltoreq.0.05) were found in nasal tissue
when comparing groups 3 (omp) and 4 (APP) vs. group 5
(challenge).
[0139] Other common respiratory and bacterial organisms were
isolated during the culturing process. Bordetella sp. was in high
numbers in all groups as well as alpha and beta streptococcal
bacteria and Pasteurella multocida. Other organisms isolated in
very low numbers were Arcanobacterium pyrogenes, E. coli,
Staphylococcus sp, Enterococcous sp, Proteus, blue fungi, and
Pastunella sp.
[0140] 4. Serology
[0141] Blood was drawn on study day 0, 7, 14, 21, 28, 34, and 41 of
the study. Western blots were performed on pooled sera from
treatment groups 1, 2, 3, and 4 on days 0 and 34 of the study to
measure immunoreactivity to the fractions. Each treatment group
consisted of three pooled groups, which consisted of 5 pigs from
that treatment group. The western blots showed detectible
seroconversion to the whole cell, supernatant, and omp fractions at
1:100 dilution, but not a 1:1000. The APP fraction had the best
reactivity and was detectable at 1:1000. For all groups, no
reactivity was found in the day 0 samples prior to treatment.
[0142] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the following claims.
REFERENCES
[0143] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0144] Monteiro, Slavic, and Michael, 2000) J Clin Microbiol 38
(10): 3759-3762 [0145] Oliveira et al, (2007) American Association
Of Swine Veterinarians, pp. 371-376. [0146] Oliveira S. (2007)
Swine respiratory bacterial pathogens: bacterial overview and
vaccine trends. In: J Wiseman, M Varley, S McOrist & B Kemp
(Eds), Paradigms in Pig Science (pp. 196-178). Nottingham, UK:
Nottingham University Press. [0147] Rullo, Papp-Szabo and Michael,
(2006) Biochimie et biologie cellulaire 84(2):184-90. [0148]
Simpson (1949) Measurement of diversity. Nature 163:688 [0149]
Slavic, et al., (2000a) Can J Vet Res. 2000 April; 64(2): 81-87.
[0150] Slavic, et al., (2000b) J Clin Microbiol. 2000 October;
38(10): 3759-3762. [0151] Versalovic et al, 1991, Nucleic Acids
Research, Vol. 19, No. 24 6823-6831
* * * * *