Multivalent inocula for lessening incidence of liver abscesses in cattle

Novel inocula for administration to ruminant animals such as cattle or sheep are provided in order to immunize the animals and lessen the incidence of liver abscesses and/or foot rot therein. In one aspect, the invention pertains to an A. pyogenes-derived vaccine including an inactivated cell culture product (e.g., cell-elaborated supernatant) from A. pyogenes cell culture in a suitable carrier. In another aspect, the invention relates to a multivalent vaccine including at least first and second bacterial components in a carrier; the first component comprises an inactivated cell culture product of A. pyogenes whereas the second component comprises an inactivated cell culture product of F. necrophorum. The inocula of the present invention find particular utility in incidences where ruminant animals are particularly subject to A. pyogenes infection leading to liver abscesses and/or foot rot, e.g., where the animals are regularly treated with an antibiotic or where cattle are fed a high grain content concentrate diet.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is broadly concerned with novel inocula for 
administration to cattle or sheep in order to lessen or prevent the 
incidence of liver abscesses and/or foot rot. More particularly, the 
invention pertains to such inocula, methods of preparing the same and 
methods of lessening the incidence of liver abscesses and/or foot rot in 
cattle or sheep via administration of the inocula. In one aspect, the 
invention relates to an A. pyogenes-derived inoculum, and in another 
aspect to a multivalent inoculum comprising a first bacterial component in 
the form of an inactivated cell culture product of A. pyogenes and a 
second bacterial component in the form of an inactivated cell culture 
product of F. necrophorum. 
2. Description of the Prior Art 
Liver abscesses in feed lot cattle are a serious economic problem, causing 
condemnation of over 3 million livers and an estimated loss of $15 million 
annually in the United States. This estimate is based primarily on 
condemnation of liver and other organs, and does not include economic 
losses stemming from reduced feed efficiencies and lowered weight gains. A 
number of studies have confirmed that cattle with abscessed livers gain 
less (average 4-5%) and have reduced feed efficiencies (average 7%) 
compared with cattle having healthy livers. The average incidence of 
abscessed liver in grain-fed cattle approximates 25-30%. 
Liver abscesses in cattle are part of a disease complex where the 
abscessation is secondary to primary foci of infection in the rumen 
epithelium. The pathogenesis can be summarized as follows: (1) ruminal 
lesions are induced by acidosis that follows rapid change in diet from 
high-roughage to high grain, prolonged feeding of high grain diet 
(sometimes referred to as an all-concentrate diet), or occasionally by 
foreign body penetration of the rumen epithelium; (2) bacteria present in 
the rumen invade the epithelium and form focal abscesses in the rumen 
wall; and (3) bacteria enter the portal circulation, and are carried to 
the liver where they localize in the parenchyma with subsequent abscess 
formation. 
F. necrophorum is the primary etiologic agent of liver abscesses in 
ruminant animals. The organism has been recognized as an animal and human 
pathogen since the late 1800s, and is associated with numerous necrotic 
disease conditions in domestic and wild animals. In addition to liver 
abscesses, the organism is also the primary etiologic agent of foot rot, 
foot abscesses, calf diphtheria, and is frequently isolated from cases of 
mastitis, metritis, and necrotic lesions of the oral cavity. 
The ability of F. necrophorum to establish in the liver is attributed to 
the production of a toxin called leukotoxin (or leucocidin). The toxin is 
soluble, proteinaceous and has specificity for bovine leukocytes. The 
leukotoxin is believed to aid in the establishment of F. necrophorum in 
the liver by directly impairing the normal defense mechanism and 
indirectly by the damage caused by cytolytic products released from 
neutrophils and macrophages to the hepatic cells. Therefore, the 
leukotoxin elaborated from F. necrophorum plays a critical role in F. 
necrophorum infection of the liver. 
F. necrophorum is a gram-negative, nonsporeforming, nonmotile, strictly 
anaerobic and pleomorphic organism. Morphologically, the organism varies 
from short rods to filamentous with pointed and rounded ends. Cell lengths 
range from coracoid bodies of 0.5-0.7 .mu.m in diameter to filaments over 
100 .mu.m. Surface colonies are 1-2 mm in diameter, circular, transparent 
to opaque, and with some strains producing .alpha. or .beta. hemolysis. 
The organism ferments glucose, fructose and maltose only weakly with final 
pH around 5.0-6.3. It ferments lactate to acetate, propionate, and 
butyrate. Butyrate is the major product from lactate fermentation. Indole 
is produced from peptone. F. necrophorum has been isolated from the normal 
flora in the oral cavity, gastrointestinal cavity, and genitourinary tract 
of humans and animals. The organism is also known to survive in the soil. 
Four biotypes (A, B, AB and C) of F. necrophorum have been described. 
Biotype A, most frequently isolated from liver abscesses, is more 
pathogenic than biotype B, which predominates in ruminal wall abscesses. 
Biotype AB is rarely isolated, and has pathogenicity intermediate that of 
biotypes A and B. Biotype C is non-pathogenic. 
It has been suggested in the past to utilize F. necrophorum bacterin as an 
agent for immunizing cattle and sheep against liver necrosis, EPO 
Application No. 460480 of Dec. 11, 1991. Specifically, virulent F. 
necrophorum isolates are inactivated using .beta.-propiolactone, followed 
by addition of adjuvants. In addition, Abe et al. (Infection and Immunity, 
13:1473-1478, 1976) grew F. necrophorum for 48 hours. Cells were obtained 
by centrifuging, washing three times with saline, and were inactivated 
with formalin (0.4% in saline). The inactivated cells were then injected 
into mice to induce immunity. Two weeks after the last booster injection, 
each mouse was challenged with viable cells of F. necrophorum. The mice 
immunized with killed cells and challenged with live cells had no 
detectable bacteria in the liver, lung or spleen for up to 28 days. It was 
concluded that immunization of mice with formalin-killed F. necrophorum 
conferred protection against infection. Garcia et al. (Canadian J. Comp. 
Med, 38:222-226, 1974) conducted field trials to evaluate the efficacy of 
alum-precipitated toxoids of F. necrophorum. The vaccine preparation 
consisted of washed cells (unlikely to contain leukotoxin) that were 
ruptured by sonication. The most promising result was achieved with the 
injection of 15.5 mg protein of cytoplasmic toxoid. In this group, the 
incidents of liver abscesses was reduced to 10% from an average 35% in the 
control group. Finally, Emery et al., (Vet. Microbiol., 12:255-268, 1986) 
prepared material by gel filtration of 18-hour culture supernate of F. 
necrophorum. This elicited significant immunity against challenged from 
viable F. necrophorum. The injected preparation contained endotoxin and 
the majority of the leukotoxic activity. 
PCT Publication WO 94/00556 published Jan. 6, 1994 describes novel F. 
necrophorum leukotoxid vaccines as well as methods of enhancing the 
elaboration of leukotoxin from F. necrophorum and of producing a 
leukotoxin vaccine. In particular, this publication discloses that F. 
necrophorum bacteria are advantageously cultured in growth media at a 
temperature of from about 35.degree.-41.degree. C. and a pH of from about 
6.5-8 for a period of from about 4-10 hours in order to maximize the 
production of leukotoxin. Thereafter, culturing is terminated and the 
leukotoxin-bearing supernate is separated and inactivated to form a 
vaccine. 
Actinomyces pyogenes is a gram-positive, cocco-bacillary shaped, 
facultative organism that is associated with a number of pyogenic 
conditions (termed "Pyobacillosis") in animals and humans. It is 
frequently isolated in mixed culture with other bacteria including F. 
necrophorum. In liver abscesses and foot rot in cattle, A. pyogenes is the 
second most frequent pathogen isolated. In addition to liver abscesses and 
foot rot, the organism is also a frequent isolated from pyogenic 
infections of a number of organs such as lungs, mammary glands, joints and 
the uterus. The pathogenic mechanism of A. pyogenes is not well 
understood. Some of the factors that contribute to pathogenicity include 
exotoxins (hemolysin or leukotoxin) and enzymes such as proteinase, 
DNases, and neuraminidase. There is also evidence that A. pyogenes in 
cattle synergistically interacts with other bacteria including F. 
necrophorum and the combination may be more virulent than individual 
species. However, no A. pyogenes-derived vaccines or inocula for 
immunizing cattle and sheep against liver abscesses and/or foot rot have 
been prepared in the past. 
SUMMARY OF THE INVENTION 
The present invention relates to inocula for cattle and sheep in order to 
immunize such ruminants and lessen the incidence therein of liver 
abscesses and/or foot rot. In addition, the invention pertains to methods 
of preparing such inocula and of lessening the incidence of the 
aforementioned pathogenic conditions in cattle and sheep. 
In one aspect of the invention, an inoculum for administration to cattle or 
sheep is provided which comprises a bacterial component and a compatible 
carrier mixed with the latter. The bacterial component consists 
essentially of an inactivated cell culture product of A. pyogenes; e.g., 
the component is selected from the group consisting of separated A. 
pyogenes cells, A. pyogenes cellular subunits or fragments, the 
supernatant elaborated by A. pyogenes cells in cell culture, and mixtures 
thereof. The carrier may be one of a number of suitable adjuvants such as 
alumina hydroxide or the oil-based or metallic salt adjuvants. 
The A. pyogenes inoculum is prepared by forming a cell culture of a strain 
of A. pyogenes in a growth media such as Brain Heart Infusion broth or 
RPMI 1640 media using an overnight culture with incubation at 
35.degree.-39.degree. C. for 12-48 hours. At the end of the incubation 
period (most preferably after about 36 hours), a cell culture product is 
inactivated using formalin, .beta.-propiolactone, heat, radiation or any 
other known method of inactivation. In particularly preferred forms, the 
entire cell culture is inactivated and chilled for two days. Thereafter, 
the inactivated cell culture is centrifuged and the supernatant antigenic 
material is recovered. As indicated previously, in alternate procedures, 
the entire cell culture, separate cells or cellular subunits may be used 
as the antigenic material. In any case, after inactivation and separation 
as desired, the antigenic material is mixed with a suitable adjuvant 
carrier. 
The invention also comprehends a multi-valent inoculum for administration 
to cattle or sheep which includes first and second bacterial components in 
a compatible carrier. The first bacterial component comprises an 
inactivated cell culture product of A. pyogenes and the second component 
comprises an inactivated cell culture product of F. necrophorum. The first 
A. pyogenes-derived bacterial component is prepared as set forth above, 
i.e., use is made of an A. pyogenes inoculum in accordance with the 
invention. The second F. necrophorum-derived bacterial component is 
prepared in the manner described in PCT Publication No. WO 94/00556 which 
is incorporated by reference herein. The multi-valent inocula of the 
invention are characterized by the property of lessening the incidence of 
liver abscesses in cattle inoculated therewith to a greater extent than 
cattle inoculated with the individual components thereof. 
In more detail, the F. necrophorum second bacterial component for the 
multi-valent vaccine is in the form of inactivated leukotoxin-containing 
supernatant elaborated by F. necrophorum cells in cell culture. Culturing 
of F. necrophorum preferably involves culturing a biotype A strain in a 
suitable growth medium such as Brain Heart Infusion broth at a temperature 
of from about 35.degree.-41.degree. C. and a pH of from about 6.5-8 for a 
period of from about 4-9 hours. The preferred strain of F. necrophorum has 
ATCC Accession No. 55329. At the end of the culturing step, leukotoxin 
supernatant is separated and inactivated by any known means, preferably 
through the use of formalin. This inactivated antigenic material can then 
be mixed with an appropriate adjuvant, of the same type described with 
reference to the A. pyogenes vaccine preparation. 
The final multivalent inoculum in accordance with the invention is prepared 
by mixing the first and second bacterial components derived from the 
respective first and second cell cultures. Preferably, the bacterial 
components are present in the final multivalent inoculum at about a 1:1 
(v/v) ratio. 
It has been determined that the inocula of the present invention will have 
particular utility in those cases where A. pyogenes becomes a significant 
etiological agent. For example, it has unexpectedly been found that where 
cattle or sheep are regularly treated (e.g., fed) with an antibiotic which 
reduces the incidence of liver abscesses and/or foot rot, a high 
percentage of infected animals exhibit A. pyogenes in their abscess 
bacterial flora. Accordingly, where ruminant animals are subjected to a 
regular regime of antibiotics such as tylosin, chlortetracycline, 
oxytetracycline, bacitracin and mixtures thereof to reduce the incidence 
of liver abscesses and/or foot rot, these animals should also be treated 
with the inocula of the present invention. In this way, the incidence of 
liver abscesses and/or foot rot will be further lessened. 
In addition, it is known that cattle fed a high concentrate diet including 
at least about 90% by weight grain characteristically have a high 
incidence of liver abscesses (greater than 50%). It has now been 
discovered that a high percentage of abscessed livers from cattle fed 
concentrate diets exhibit A. pyogenes upon bacteriological examination. 
Therefore, cattle fed concentrate diets can also materially benefit from 
treatment with the inocula of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The following examples describe the preferred techniques for the production 
and use of a monovalent A. pyogenes-derived inoculum and of a multivalent 
inoculum containing A. pyogenes and F. necrophorum antigenic components. 
It is to be understood, however, that these examples are presented by way 
of illustration only, and nothing therein should be taken as a limitation 
upon the overall scope of the invention. 
EXAMPLE 1 
In this example, the bacterial flora of liver abscesses from feedlot cattle 
fed with or without the antibiotic tylosin were examined. A total of 36 
liver abscesses from tylosin-fed cattle were examined, along with 41 liver 
abscesses from cattle not fed tylosin. This study revealed that there was 
a higher incidence of A. pyogenes in the liver abscesses of tylosin-fed 
animals. Accordingly, it was deduced that in tylosin-fed animals, the role 
of F. necrophorum is lessened and A. pyogenes assumes a greater role than 
in animals not fed tylosin. The results of this study are set forth in the 
following table. 
TABLE 1 
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Bacterial flora of liver abscesses in cattle fed tylosin or no tylosin 
Bacteria Tylosin No Tylosin 
______________________________________ 
No. of abscesses cultured 
36 41 
F. necrophorum 36/36 (100).sup.a 
41/41 (100) 
Subsp. necrophorum.sup.b (biotype A) 
18/36 (50) 
34/41 (82) 
Subsp. funduliforme (biotype B) 
3/36 1/41 
Actinomyces pyogenes.sup.b 
19/36 (53) 
4/41 (100 
______________________________________ 
.sup.a Numbers in parentheses are percentages 
.sup.b Chi Square test P &lt; .01 
Although the origin of F. necrophorum found in liver abscesses is well 
known, the source of A. pyogenes is not understood. Because A. pyogenes is 
aerobic, it is not a normal inhabitant of the rumen; however, the 
bacterium may be a component of the epimural flora of the rumen (bacteria 
attached to the rumen wall). Facultative or aerobic bacteria have a better 
chance of surviving in anaerobic conditions by adhering to the rumen wall 
where extensive blood supply provides oxygen. The close proximity of the 
rumen wall enhances the opportunity of the bacterium to get into portal 
circulation and thereby enter the liver. The bacteria could remain dormant 
and multiply if conditions (such as entry into the liver) become 
conducive. The higher incidence of A. pyogenes in tylosin-fed cattle is 
surprising because the organism is quite sensitive to tylosin. 
EXAMPLE 2 
In this example, the liver abscesses of cattle fed an all-concentrate diet 
(100% grain) were bacteriologically examined. It is known that feedlots 
making use of all-concentrate diets have a high incidence of liver 
abscesses (greater than 50%). In this study, the livers of 24 animals were 
examined and it was found that 19 were abscessed. Eighteen of these 
abscesses were cultured and F. necrophorum was found in 12 abscesses 
whereas A. pyogenes was found in 17 abscesses. This data is summarized in 
the following Table 2. 
TABLE 2 
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Bacterial flora of liver abscesses in cattle fed all concentrated diet 
Bacteria Incidence 
Percentage 
______________________________________ 
No. of animals 24 
Abscessed 19 79 
No. of abscesses cultured 
18 
F. necrophorum 12 67 
Actinomyces pyogenes 
17 94 
______________________________________ 
In the basis of the tests sets forth in Examples 1 and 2, it is believed 
that there is a synergistic interaction between F. necrophorum and A. 
pyogenes in causing liver abscesses. The presence of A. pyogenes may 
enhance the virulence of F. necrophorum or vice-versa. Table 3 sets forth 
the characteristics of the respective bacteria and their interactions. 
TABLE 3 
______________________________________ 
Fusobacterium 
Actinomyces 
Characteristics 
necrophorum pyogenes Interaction 
______________________________________ 
O.sub.2 relationship 
Anaerobic Facultative 
A. pyogenes utilizes 
O.sub.2 to create anaerobic 
condition for F. 
necrophorum growth 
Substrate and 
Ferments lac- 
Produces A. pyogenes provides 
endproducts 
tate lactate substrate for F. necro- 
phorum growth 
Leukotoxin 
Strongly posi- 
Negative or 
Leukotoxin of F. 
production 
tive weakly posi- 
necrophorum protects 
tive against phagocytosis 
Hemolysis 
Weakly positive 
Strongly Hemolytic activity of 
positive A. pyogenes provides 
iron required for F. 
necrophorum growth 
______________________________________ 
EXAMPLE 3 
In this example, the preparation of a monovalent A. pyogenes vaccine is 
described, as well as the preparation of a multivalent A. pyogenes/F. 
necrophorum vaccine. 
A. pyogenes Vaccine 
A suitable strain of A. pyogenes is grown in a Brain Heart Infusion broth 
(DIFCO Laboratories, Detroit, Mich.) or RPMI 1640 medium (GIBCO 
Laboratories, Grand Island, N.Y.). The growth medium is inoculated with an 
overnight culture (1-5% inoculum size) of A. pyogenes and is incubated at 
35.degree.-39.degree. C. for 12-48 hours in a 5% CO.sub.2 atmosphere (a 
standard atmosphere could also be used). At the end of the incubation 
period, the culture is inactivated by adding formalin (0.3-0.4%) on a 
vol./vol. basis. The inactivated whole culture is chilled in an ice bath 
and refrigerated for one or two days. Thereafter, the inactivated cell 
culture is centrifuged (13,500 g for 15-30 minutes) and the supernatant 
antigenic material is recovered. In alternative procedures, the whole cell 
culture (bacterin) or cellular subunits can be used as the antigenic 
material. 
The antigenic material is then mixed with a suitable adjuvant (e.g., 
aluminum hydroxide or other commercially available oil-based or metallic 
salt adjuvant) to complete the vaccine. The vaccine may then be 
conventionally administered to sheep or cattle (one or more vaccinations) 
to elicit antibodies in the animals and will prevent the establishment of 
A. pyogenes in the animal's liver or feet, or any other organs. 
A. pyogenes/F. necrophorum Vaccine 
The F. necrophorum leukotoxoid vaccine component is prepared by growing a 
F. necrophorum biotype A strain (e.g., strain 25, ATCC Accession No. 
55329) in a pre-reduced (0.05% cysteine HCl) anaerobically sterilized 
Brain-Heart Infusion broth (DIFCO Laboratories, Detroit, Mich.) at 
39.degree. C. for 7 hours (absorbance 0.8 at 600 nm). The culture 
supernatant is obtained by centrifugation at 13,500 g for 30 minutes at 
4.degree. C., filtered through a 0.45 .mu.m membrane filter (Micron 
Separations, Inc., Westborough, Mass.) and inactivated with 0.3% formalin. 
F. necrophorum leukotoxin and protein concentrations in the cell culture 
supernatant are assayed before and after inactivation with formalin. 
The inactivated supernatant is mixed with Ribi adjuvant (premixed, sterile 
form from Ribi Immunochem Research, Inc., Hamilton, Mont.) or other 
adjuvant as described above at a level of 90% leukotoxin supernatant/10% 
adjuvant. The mixture is then emulsified in a homogenizer. 
In order to create the multivalent vaccine, the A. pyogenes vaccine and the 
F. necrophorum vaccine component are mixed on a 1:1 v/v basis. The 
multivalent vaccine can then be conventionally administered (typically via 
one or more injections) to cattle or sheep. 
The monovalent or multivalent vaccines of the invention may be 
conventionally administrated via parenteral injection or other known 
techniques in order to elicit the production of appropriate antibodies in 
the ruminant animals. Such administrations may be at one time or multiple 
administrations spaced over a period of time.