A sub-cellular antigenic fraction of Bordetella bronchiseptica protects against the incidence and severity of swine atropic rhinitis.

BACKGROUND OF THE INVENTION 
Atropic rhinitis is prevalent in various countries of the world and causes 
severe economic losses in the swine-raising industry in terms of both 
inhibition of growth and reduction of feed efficiency. The causative agent 
for this disease has been shown to be B. bronchiseptica. At least 1.8 
million cases occur in the U.S. alone each year and entire herds may 
become infected before clinical signs appear. Once apparent, the effect in 
terms of morbitity is essentially irreversible despite the use of 
antibiotics. Therefore, prophylaxis rather than cure is the only way to 
control this disease. 
OBJECTS OF THE INVENTION 
It is an object of the present invention to provide a safe and effective 
vaccine to prevent atrophic rhinitis. Another object is to provide a 
sub-cellular vaccine to eliminate the incedence and severity of atrophic 
rhinitis in swine. A further object is to provide a biochemically 
characterized sub-cellular vaccine which protects swine against atropic 
rhinitis. Still another object is to provide formulations for 
administering this vaccine. Another object is to provide a method for 
preparing this vaccine. Yet another object is to provide methods of 
administering this vaccine. These and other objects of the present 
invention will be apparent from the following description. 
SUMMARY OF THE INVENTION 
A sub-cellular vaccine effective against atropic rhinitis isolated from a 
cell wall antigenic fraction of a pathogenic isolate of B. bronchiseptica 
chemically characterized in containing from about 53 to about 57% protein 
(Lowry), from about 10 to 13% carbohydrate (of which about 19% is hexose 
and about 19% hexosamine), from about 1 to about 1.4% sialic acid, and 
about 0.18% 2-keto-3-deoxyoctanate (KDO). 
DETAILED DESCRIPTION 
The present invention relates to the isolation and purification of a cell 
wall fraction of a pathogenic isolate of B. bronchiseptica. The cell wall 
fraction is obtained from a killed cell paste of B. bronchiseptica. The 
cells may be killed by suitable means, e.g., chemical treatment. The 
killed cell paste is homogenized, e.g., by high speed mixing, extracted in 
an aqueous medium and centrifuged. The homogenization takes place in the 
presence of a salt of a strong acid and a strong base, e.g., LiCl, at a 
concentration of from about 0.05 M to about 1 M, or in the presence of a 
nonionic detergent such as alkyl phenoxy polyethoxy ethanol (Triton X-100) 
or an anionic detergent such as sodium deoxycholate. The detergent is 
employed at a concentration of from about 0.5 to about 10%. The treatment 
with the salt or detergent disrupts ionic bonds and salt bridges which 
maintain the integrity of the bacterial membrane and cell wall. Treatment 
with salt is preferred. A product of enhanced purity is obtained, although 
in reduced yield, by a combined treatment using salt and detergent 
sequentially. The extraction may take place at room temperature or at 
lowered temperatures down to about 4.degree. C. or at elevated 
temperatures up to about 75.degree. C. In general extraction times are 
reduced at elevated temperatures. The centrifugation takes place at from 
about 12,000 XG to about 20,000 XG for from about 0.5 hour to about 3 
hours. In general centrifugation time at higher G force is reduced. The 
supernatant fluid resulting from the centrifugation is treated with RNase 
and DNase under conditions effective to digest nucleic acids, and dialyzed 
to remove nucleic acid fragments and to yield the desired antigenic 
fraction. The dialyzed antigen may be lyophilized for storage. 
Alternatively the dialysed product may be subjected to column 
chromatography, e.g., Sepharose 4B, Biogel 1.5 M or 5 M, and the like, 
whereby the first peak is collected and used as the desired antigenic 
fraction. 
The cell wall antigenic fraction of the present invention is immunogenic 
and protects both mice and swine against both homologous and heterologous 
challenge with a pathogenic isolate of B. bronchiseptica field isolates. 
The sub-cellular cell wall antigenic fraction of a pathogenic isolate of 
B. bronchiseptica obtained according to the foregoing method is chemically 
characterized in containing at least about 53% Lowry protein, typically 
from about 53 to about 57% Lowry protein, from about 10 to about 13% 
carbohydrate (of which about 19% is hexose, and about 19% is hexosamine), 
from about 1 to about 1.4% sialic acid, and about 0.18% KDO. 
The cell wall antigenic fraction of the present invention may be sterilized 
by filtration or treatment by chemicals such as thimerosol, phenol, 
formaldehyde and the like, and subdivided into a suitable container for 
distribution and administration as a vaccine. It may be administered in a 
suitable physiologically acceptable medium such as, for example, water for 
injection, saline, phosphate buffered saline, and the like. It may be 
combined with adjuvants such as, for example, those disclosed in U.S. Pat. 
No. 3,983,228 or with carriers, e.g., alum.

The following examples illustrate the present invention without, however, 
limiting the same thereto. 
EXAMPLE 1 
A cell paste of B. bronchiseptica killed with thimerosal and harvested by 
Sharples centrifugation is fractionated to obtain and characterize 
protective vaccine cell wall fractions. Initially, the wet cell paste is 
homogenized with 0.2 M LiCl in an electric blender with overhead blades at 
a concentration of 20 ml/gram of wet paste. This mixture is then extracted 
in a shaking water bath for 2 hours at 45.degree. C., and subsequently 
centrifuged at 16,000.times.G for 60 minutes at 20.degree. C. The 
supernatant fluid is made 1.0 mM in MgCl.sub.2, brought to 37.degree. C. 
and digested for 45 minutes with pancreatic RNAse and DNAse at 
concentrations of 10 .mu.g/ml each. The mixture is then dialyzed against 
H.sub.2 O and lyophilized. All fractions are tested for protective 
activity as explained below. Both the extract and the residue are active; 
a second extraction of the cell paste, however, removes the residual 
activity. The lyophilized extract is chromatographed on a BioGel 5 M 
column and fractionated into numerous sub-fractions; however, only one of 
these (eluting at 1 V.sub.o) is protective. This material although 
substantially enriched in protective immunogen is neither immunologically 
nor chemically homogeneous. It is characterized chemically as having the 
following analysis: 
______________________________________ 
Assay 
______________________________________ 
Protein Lowry 54.4% 
Hexoses Anthrone 2.45 
Carbohydrate Phenol-H.sub.2 SO.sub.4 
11.5 
1.6 
Hexosamine Berman-Gatt 2.5 
Sialic Acid Warren 1.2 
KDO Osborn 0.18 
______________________________________ 
This fraction protects mice, at dosed of 1-100 .mu.g, not only against 
homologous challenge, but also against challenge with heterologous B. 
bronchiseptica isolates. 
EXAMPLE 2 
Fourteen groups of mice (10 animals per group) are injected 
intraperitoneally (I.P.) with 0.1 ml of a PBS (phosphate buffered saline) 
solution containing 100 .mu.g of the product of Example 1 or with 0.1 ml 
of PBS only. Seven to fourteen days post immunization, the mice are 
challenged with varying amounts of differing strains of B. bronchiseptica 
(I.P. injection in 1.0 ml 7.5% Hog Gastric Mucin/mouse). The mice are 
observed daily and the percentage of survivors determined after 3 days. 
The following results are obtained: 
______________________________________ 
% Survivors 
Challenge No. of No 100 .mu.g 
Group Strain Bacteria Antigen Antigen 
______________________________________ 
I Homologous 10.sup.4 10 90 
II Homologous 10.sup.6 0 80 
III MO-2 10.sup.3 100 100 
IV MO-2 10.sup.4 100 100 
V MO-2 10.sup.5 30 100 
VI MO-2 10.sup.6 0 100 
VII SD12972 10.sup.3 100 100 
VIII SD12972 10.sup.4 80 100 
IX SD12972 10.sup.5 80 100 
X SD12972 10.sup.6 10 100 
XI ILL4673 10.sup.3 100 100 
XII ILL4673 10.sup.4 90 100 
XIII ILL4673 10.sup.5 55 100 
XIV ILL4673 10.sup.6 10 90 
______________________________________ 
EXAMPLE 3 
Four groups of pigs (16 animals per group) infected with B. bronchiseptica 
and one group of 16 uninfected pigs are injected as indicated in the 
following table. 
______________________________________ 
Material Injected 
Group Rhinitis Score 
______________________________________ 
1 (Uninfected) 
PBS, no antigen (control) 
2.69 
2 (Infected) 
Product of Example 1, 
PBS and Adjuvet 1.94 
3 (Infected) 
Product of Example 1, 
Adjuvet (2X) 2.50 
4 (Infected) 
Product of Example 1, 
Burns-Biotec (2X), PBS 
5.19 
5 (Infected) 
PBS, no antigen (control) 
8.53 
______________________________________ 
EXAMPLE 4 
The procedure of Example 3 is repeated with another four groups of pigs (16 
animals per group ) infected with B. bronchiseptica and one group of 16 
uninfected pigs are injected as indicated in the following table. 
______________________________________ 
Material Injected 
Group Rhinitis Score 
______________________________________ 
1 (Uninfected) 
PBS, no antigen (control) 
3.46 
2 (Infected) 
Product of Example 1, 
PBS and Adjuvet 3.08 
3 (Infected) 
Product of Example 1, 
PBS (2X) 4.93 
4 (Infected) 
Product of Example 1, 
Alum (2X) 4.86 
5 (Infected) 
PBS, no antigen (control) 
8.31 
______________________________________ 
EXAMPLE 5 
A portion of the final product of Example 1 (10 mg) is added with stirring 
to 2 ml of water, and 1 ml of a 25% suspension of a mixture of Al.sub.2 
O.sub.3 and Al(OH.sub.3) (alum), and 5 ml of water. Over 90% of the Lowry 
protein is adsorbed to the alum. 
EXAMPLE 6 
A portion of the final product of Example 1 (25 mg) is added to 10 ml of 
PBS. While stirring 0.1 N NaOH is added slowly to adjust the pH to 6.8. 
Mixing is continued for 1 hour at room temperature. The solution is then 
centrifuged at 1,500.times.G for 10 minutes. The supernatant liquid is 
decanted and the pellet is resuspended with saline solution to the 
original volume (10 ml). 
EXAMPLE 7 
A portion of the final product of Example 1 (25 mg) is added to a mixture 
of 5 ml of a peanut oil adjuvant prepared as described in Example 5 of 
U.S. Pat. No. 4,069,313 and 5 ml of PBS with agitation until a uniform 
mixture is obtained. 
EXAMPLE 8 
The procedure of Example 1 is repeated except that the cell paste is 
extracted with 2% Triton X-100 for 30 minutes at room temperature rather 
than with LiCl. The yield is 35% of that obtained with LiCl and on an 
equal weight basis the antigenic product is only about 20% as effective as 
that obtained with LiCl. 
EXAMPLE 9 
The final product of Example 1 is further purified by extraction with 5% 
Triton X-100 for one hour at room temperature. This treatment removes most 
of the endotoxin contaminant although the protective antigen is recovered 
only in about 10% yield. 
EXAMPLE 10 
A cell paste prepared as described in Example 1 is homogenized by pressure 
(15,000 psi, 1,020 atmospheres). The cell walls are separated from 
cytoplasmic fluid and unbroken cells by differential centrifugation. The 
cell walls are washed with and extracted with sodium deoxycholate (1%). 
The extract is separated from the cell wall residue, precipitated 
sequentially with 50% ammonium sulfate and 80% ethanol, dialyzed and 
lyophilized. The lyophilized product is as protective to mice against a 
lethal dose of B. bronchiseptica as the LiCl extracted product but purer 
as it contains a higher percentage of protein and less lipopolysaccharide. 
The yield, however, is very low (about 5%) compared to the use of LiCl. 
EXAMPLE 11 
The final product of Example 1 is further fractionated on Sephadex G-100 
columns to remove the lipopolysaccharides. The gel filtration on Sephadex 
G-100 is carried out in the presence of 0.5% sodium deoxycholate, 1 mM 
ethylenediaminetetraacetic acid, and 0.05 glycine, pH 10. In preparation 
for chromatography, the samples are resuspended in 10 mg/ml in the above 
buffer, except that the sodium deoxycholate concentration is 1% and the pH 
is 11.5. These suspensions are centrifuged at 100,000.times.G for 2 hours 
and the clear supernatant fluids are applied to G-100 columns. The column 
fractions are monitored for refractive index and optical density at 260 
and 280 m.mu.; fractions are also assayed for KDO and carbohydrate 
(phenol-sulfuric acid assay). The clearly resolved peaks are pooled, made 
0.5 M in NaCl and 80% in ethanol. The resulting precipitates are collected 
by centrifugation, washed with ethanol and acetone and dried over 
CaCl.sub.2 in vacuo at 4.degree. C. The individual peaks are chemically 
characterized. The Vo peak of G-100 column is found to be protective 
against a lethal dose of B. bronchiseptica in the hog gastric mucin mouse 
model.