Fowl Cholera in Turkeys: The Efficacy of Adjuvant Bacterins

Author(s): Jagmohan L. Bhasin and Ernst L. Biberstein

Source: Avian Diseases, Vol. 12, No. 1 (Feb., 1968), pp. 159-168
Published by: American Association of Avian Pathologists
Stable URL: http://www.jstor.org/stable/1588097 .
Accessed: 23/06/2014 05:56

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .
http://www.jstor.org/page/info/about/policies/terms.jsp

.
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org.

American Association of Avian Pathologists is collaborating with JSTOR to digitize, preserve and extend
access to Avian Diseases.

http://www.jstor.org

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
 FOWL CHOLERA IN TURKEYS
THE EFFICACY OF ADJUVANT BACTERINS"
Jagmohan L. Bhasin and Ernst L. Biberstein
Department of Veterinary Microbiology
School of Veterinary Medicine
University of California, Davis

Received 19 June 1967

INTRODUCTION
Ever since the production of the classical attenuated fowl
cholera vaccine by Pasteur, many attempts have been made to
prevent outbreaks of fowl cholera in chickens. His method could
not be duplicated successfully, however, and a search for a more
dependable method in controlling the disease effectively has been
conducted since the early 1920's. The effectiveness of earlier
aqueous avirulent vaccines (7) and bacterins (13) has always been
variable (8,19,9,10).
Carter (2) compared the immune response in mice to bac-
terins made from broth cultures and chicken embryo vaccines made
from Pasteurella multocida, type A. Chicken embryo vaccine was
much superior to broth-derived bacterin in immunizing value.
Bierer et al. (1) described a phenol-killed autogenous bacterin
which afforded some protection to turkeys in an infected flock.
The challenge, however, was carried out within 20 days of vac-
cination.
The introduction of adjuvants has considerably improved the
efficacy of killed bacterins, although the side effects of these are
not very well understood (5). Freund (6), while reviewing the lit-
erature on adjuvants, emphasized the effectiveness of light mineral
oil in water-in-oil emulsions for the stimulation of a high degree
of immunity and prolonged antibody response to bacterins.
Heddleston and Reisinger (10) first showed that a single dose
of a bacterin consisting of a water-in-oil emulsion of formalinized
P. multocida was capable of producing a high degree of immunity
against fowl cholera in chickens for at least 9 months. Alum-pre-
cipitated vaccine, chick embryo vaccine, and other commercially

ASupported in part by USPHS General Research Support Grant FR-05457

and a grant by the California Turkey Federation.

159

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
160 Jagmohan L. Bhasin and Ernst L. Biberstein

prepared bacterins were of questionable immunizing value accord-

ing to their study.
Priestley (16) stressed the importance of using highly cap-
sulated strains for bacterin production. A very definite criterion
for selecting vaccine strains was also outlined by Heddleston and
Hall (9).
Several methods have been used to kill bacterial suspensions
for bacterin production. Priestley (16) emphasized that heat-killed
suspensions of highly capsulated strains were most immunogenic
if heated at not more than 56?C for 30 minutes. Chemical agents
such as formalin have been used widely. An oil-emulsified formalin-
killed bacterin that protects chickens against fowl cholera for at
least 54 weeks has been prepared by Heddleston and Reisinger
(10). Chute et al. (4) employed formalin and beta-propiolactone
to kill bacterial cultures before suspending them in adjuvants.
Ethylene oxide (9) has also been used to inactivate bacterial sus-
pensions.
Although many commercially produced bacterins have been
used in turkeys in an attempt to prevent P. multocida infection,
little has been done to evaluate their effectiveness. This paper
presents the results of a study to determine the effectiveness of
different bacterins in controlling experimental fowl cholera infec-
tion.
MATERIALS AND METHODS
Turkeys. Three hundred and forty white broad-breasted tur-
keys raised on a commercial breeding farm in California were
used. At the time of vaccination they were 12 weeks old. There
was no history of fowl cholera outbreaks on that particular farm,
and no fowl cholera bacterins had been used. No detectable hemag-
glutinating (HA) antibodies to P. multocida, types A, B, and D,
were detected in a randomized sample taken from 45 of the birds
in question. Nasal-cleft swabs from the same birds cultured on
blood agar were also negative for Pasteurella organisms.
The birds were housed in two 26 X 50-foot sheltered pens at
the Experimental Tract of the School of Veterinary Medicine, and
usual poultry facilities were provided toward their care and man-
agement.
Mice. Adult Swiss mice were used.
Cultures:The strains of P. multocida (stock 19) used for bac-
terin production and (stock 19 and 26) for challenge had been re-

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
 Fowl cholera in turkeys 161

cently isolated from an acute fowl cholera outbreak in turkeys in

California. The two isolates were identified biochemically as P.
multocida, and serologically as type A by Carter's method. Both
were highly pathogenic for adult Swiss mice and 12-week-old tur-
keys. They were held in the lyophilized state until further use.
Bacterinpreparation. The lyophilized strain of P. multocida
(stock 19) was reconstituted in physiological saline solution and
streaked on yeast-proteose-L-cysteine (YPC) agar plates (15).
Plates were incubated for 18 hr, and a single smooth iridescent
colony showing encapsulated cells (14) was transferred to 100 ml
of brain heart infusion broth. After 6-8 hr of incubation on a low-
speed shaker (Brunswick), 3 ml of this broth culture was seeded
on YPC agar in Roux bottles and incubated for 18 hr. Then the
growth was washed off in 15 ml of saline. Glass beads were used
to remove the cells from the surface of the agar. Washings from
several bottles were pooled and kept at 4?C for short periods pend-
ing further processing on the same day.
Formalin or heat treatment was used to kill the bacterial sus-
pensions. Formalin was added to the bacterial suspensions to make
a final concentration of 0.25%. Heat treatment was done by heat-
ing 50-ml amounts of the suspension in 250-ml flasks containing
glass beads. The flasks were immersed in a water bath at 56-57?C
for 30 min and shaken 3 or 4 times to ensure uniform exposure of
all cells. Both formalin-killed and heat-killed suspensions were held
in the refrigerator overnight and their sterility was checked by the
usual bacteriological methods and pathogenicity tests in mice. The
suspensions were then standardized spectrophotometrically to an
optical density comparable to MacFarland tube No. 10 (9).
Two bacterins were prepared from each suspension by either
mixing three volumes of the suspension and one volume of alum-
inum hydroxide (Amphojel, unflavored, Wyeth Company) or emul-
sifying in a Teflon blender one volume of the suspension in one
volume of complete Freund's adjuvant (Difco Laboratories). The
final products (Table 1), labeled Bacterin I and II (formalin-
killed) and Bacterin III and IV (heat-killed), were held at refrig-
erator temperature while their sterility and safety were confirmed
by culture and pathogenicity tests in turkeys and mice.
Vaccination. Four groups of 70 birds each were identified by
wing band numbers. Groups 1 and 2, which respectively received
Bacterins I and II, were confined together in one pen, while groups
3 and 4, which received Bacterins III and IV, were confined to the

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
162 Jagmohan L. Bhasin and Ernst L. Biberstein

second pen. The birds were inoculated with 0.5 ml of the respective
bacterin subcutaneously in the side of the neck. Group 5, contain-
ing 30 birds, served as an unvaccinated control and was allowed
to mix in both the pens with the vaccinated birds.
Challenge inoculum. Bacteria for challenging were prepared by
transferring an 18-hour-old smooth iridescent colony from YPC
agar plate to 100 ml of brain heart infusion broth. After eight
hours of incubation the cell suspension was standardized to contain
approximately 2.7 X 10'9cells per ml.
Challenge. The immunity of the birds was challenged 4, 8, 12,
and 16 weeks after vaccination. Twenty birds from each vaccinat-
ed group and five unvaccinated controls were selected randomly
and transferred to an aseptic unit in the specific pathogen-free
laboratory. The birds were challenged by swabbing the nasal cleft
with a cotton swab saturated with the challenge inoculum. All
birds were observed for 7 days and the survivors then killed. Swabs
from the nasal cleft, trachea, and liver were cultivated on blood
agar for Pasteurella organisms. Any dead or visibly sick birds were
also cultured. Birds showing paralytic symptoms were sacrificed
on the seventh day, and the brain also was cultured.
Serology. On the day of challenge 5 ml of blood was taken from
the large brachial vein of each vaccinated and control bird. The
serum obtained was either tested within 24 hr of collection or
stored in the frozen state for later testing. All serum samples were
tested for HA antibodies. The technique of the HA test was similar
to that described by Carter (3).
Passive immunity tests. Four serum batches having HA titers
of 1:64, 1:128, 1:256, and 1:512 were used in the mouse protec-
tion test (18). Each batch consisted of ten samples. Four mice
were injected subcutaneously with 0.2 ml of a serum sample and
after 24 hr were inoculated with 0.1 ml of a 10-5 dilution of an 8-hr
broth culture. An equal number of controls (unprotected) were
similarly inoculated with the challenge inoculum. Mice were ob-
served for 3 days after challenge.
RESULTS
The bacterins used were tolerated well by the vaccinated birds.
Six birds were lost prior to challenge, from causes unrelated to
Pasteurella infection. One of the birds was paralyzed, and the oth-
ers were killed because of broken wings or legs.
All bacterins used conferred significant degrees of immunity

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
 Fowl cholera in turkeys 163

Table 1. Comparative results of the efficacy of bacterins as judged by

challenge.'

Bacterin Weeks postvaccination

Inactivating Adjuvant Challenge 4 8 12 16

agent
Formalin I Aluminum S/C 6/20 5/20 4/20 4/10
hydroxide
II Freund's S/C 5/20 6/20 6/20 3/10

56?C/30 min II: Aluminum S/C 4/20 2/20 1/20 0/5

hydroxide
IV Freund's S/C 3/20 2/20 1/20 2/11

Unvaccinated controls S/0 5/5 5/5 5/5 5/5

AChallenged by swabbing nasal clefts with culture.

S = number of birds visibly sick.
C = number of birds challenged.

to a challenge which invariably killed 100% of the controls (Table

1). The responses to the two challenging strains, which, as indi-
cated, originated from the same outbreak, were essentially iden-
tical, so they are tabulated jointly.
Table 1 indicates a significant difference in protective abili-
ties between heat-killed and formalinized bacterins, with the former
much superior. This difference became even more pronounced with
the passage of time. At four weeks the immune status of the birds
in both groups was essentially similar, but at eight weeks and
thereafter until the end of the experiment the rate of mortality
(Table 1) was reduced more impressively in the group of birds
which had received heat-killed bacterins. In fact, the mortality con-
tinued to decline in birds immunized with Bacterin III (heat-killed,
Aluminum hydroxide) after it had reached its lowest point in the
other groups and had shown a rise at the last challenge. No signifi-
cant difference was observed in the ability of the two heat-inacti-
vated bacterins to establish immunity, although Bacterin IV in
Freund's adjuvant appeared slightly less protective, especially in
the light of the last challenge.
All the deaths in unvaccinated controls occurred within 24 hr.
These birds were of the same age and had been raised along with
the immunized groups. Generally, the incubation period of the ex-
perimental disease was much shorter (12-18 hr) in 12-week-old
controls than in 20- or 28-week-old birds (24 hr). Irrespective of
the incubation period, however, once the signs of the disease de-
veloped, death generally followed within 18-24 hr.
Deaths in vaccinated groups usually occurred late (in about
72 hours), and in no case were peracute deaths observed. Sick birds

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
164 Jagmohan L. Bhasin and Ernst L. Biberstein

Table 2. Results of Hemagglutination tests.

4 weeks 8 weeks 12 weeks 16 weeks

Bacterin No. of HA titerA No. of HA titer No. of HA titer

No. of HA titer
turkeys turkeys turkeys turkeys
vaccinated 1 2 3 4 vaccinated 1 2 3 4 vaccinated 1 2 3 4 vaccinated 1 2 3 4

I 20 3B 6 7 4 20 4 7 9 - 20 7 3 9 1 10 3 7 - -
II 20 1 5 8 6 20 4 4 7 3 ) 2 1 7 9 10 1 6 3-
III 20 5 11 4 - 20 3 10 7 - 20 1 9 8 7 - 3 2 -
IV 20 3 5 6 6 20 2 8 6 4 20 4 3 5 8 11 25 4 -

A1 = 1:64, 2 = 1:128, 3 - 1:256. 4 = 1:512.

"Number positive for the titer.

died on the fourth or fifth day after challenge or survived to the

seventh day, when they were killed. Visibly sick birds are recorded
in Table 1.
Following the challenge, paralytic signs were observed in birds
immunized with oil-adjuvant bacterins (both heat-inactivated and
formalinized). A total of five birds developed torticollis after four
days. Pasteurella organisms were localized in brain tissue in all five
of the birds. None of these birds had septicemia, for the organ-
isms were absent from heart blood, spleen and liver. The trachea
and nasopharynx, however, yielded almost pure cultures of P.
multocida.
Based on the recovery of organisms from the vaccinated birds
after challenge, heat-killed bacterins were, again, most effective
(Fig. 1). Isolations of the organisms were much less frequent from
birds immunized with heat-inactivated aluminum-hydroxide-ad-
sorbed bacterin.
Antibody response (Table 2) as demonstrated by HA anti-
bodies lacked a consistent pattern. The four groups of birds im-
munized with four different bacterins had essentially a similar
range of HA titers. No correlation was observed between HA
titers and protection. Birds showing a titer of 1:64 were still able
to resist challenge, while some of those with higher titers suc-
cumbed.
No passive immunization of adult mice could be achieved by
injection of immune turkeys' serums. All mice died when challenged
with 0.1 ml of 10-5 dilutions of Pasteurella organisms.
DISCUSSION
The apparent superiority of the heat-killed adjuvant bacterin
in establishing a high degree of immunity in turkeys is of interest,
particularly in light of the essential similarity of HA titers in all

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
 Fowl cholera in turkeys 165

birds regardless of immunizing technique. It is probably attribu-

table to the nature of the treatment of the bacterial suspensions.
The formalinization procedures used in the present study required
a concentration of 0.25% formalin. The time for its action allowed
to ensure proper killing of the cells was at least 18 hours. An ex-
posure of this duration may well have altered the antigenic and im-
munogenic properties. It is conceivable that the protective antigens
are altered to some degree whereas capsular antigens retain their
integrity. The above discrepancies were also noted by Heddleston
and Hall (9). In their results, rate of survival of immunized birds

NASAL CLEFT
70
70-
60-
i1 ill

30-
20-
5-
WEEKS. 4 12 4812 481216 481216
8 16 8 16 8 16 8 16

60-'-
TRACHEA
45- -

30- _
15-

,. rvo .. . .. .
.....i I II
W ttC
r.i
481z6 4812 6 48 126 481216

WEEKS- 4 2
8 16 8 16 8 16 '8-16
BACT.I BACT.
I BACT.I BACT.11
Fig. 1. Recovery of P. multocida from immunized birds after challenge
Weeks - time interval between immunization and challenge.

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
166 Jagmohan L. Bhasin and Ernst L. Biberstein

was higher for ethylene-oxide-killed bacterin than for formalin-

killed bacterins. Rebers et al. (17) recently showed that formalin-
ized extracts of a noncapsulated avirulent laboratory mutant strain
of P. multocida were equal to or better than extracts from cap-
sulated virulent strains in immunizing chickens. Purified prepara-
tions of extracts of this avirulent strain actively immunized mice
and chickens against challenge with virulent organisms. The ser-
ums of immunized rabbits gave specific reactions with the homolo-
gous antigen in Ouchterlony plates and agglutinated bacterial cells
of the same strain. These observations are in harmony with our
conclusions that the protection-inducing antigens are not confined
to the capsule.
Very little has been published regarding the effect of mild
heat on Pasteurella antigens. Priestly (16), from experiments on
the thermal inactivation of organisms, concluded that a highly effi-
cient vaccine can be prepared provided the capsulated virulent
strains of P. multocida are heated at not more than 56?C for 30
minutes. There is no explanation, however, for the better immuniz-
ing ability of heat-killed bacterins.
The use of different adjuvants is well established in prolong-
ing retention, thereby slowing the release of an antigen from an
injected site. The question of whether oil-adjuvant bacterins are
better than aluminum-hydroxide-adsorbed bacterin has not been
clearly answered. Heddleston and Reisinger (11) concluded that al-
uminum-hydroxide-adsorbed bacterins did not stimulate a higher
degree of immunity than oil-adjuvant preparations. Our finding
indicates that aluminum-hydroxide-adsorbed bacterins were equal
to, or even slightly more effective than, oil-adjuvant bacterins (see
Table 1). It is of interest that the paralytic syndrome with localiza-
tion of Pasteurella organisms in the brain, observed in immune
birds after challenge, was restricted to the group immunized with
oil-adjuvant bacterin. It remains open to speculation whether this
syndrome or the apparent tissue predilection of the organism in
the brain was due to a lesser degree of protection conferred by these
bacterins or to a specific peculiarity of the oil-adjuvant bacterin.
In any case, the absence of bacteria in the blood, spleen, and liver
argues in favor of local extension from the nasopharynx as being
the mechanism responsible for this involvement.
The 100% mortality of unvaccinated controls indicates the
severity of the challenge inoculum. It was noted, however, that
older birds (20-28 weeks) had a longer incubation period than

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
 Fowl cholera in turkeys 167

younger birds (12 weeks). This apparent difference in susceptibil-

ity may well have been due to the development of partial age im-
munity.
The lack of correlation between antibody response and im-
munity in fowl cholera has been noted by others. Heddleston and
Watko (12), for example, indicated that there was no clear-cut
relation between agglutination titers and immunity in chickens ex-
posed to homologous challenge. Forty-three percent of turkeys and
77% of chickens with no agglutinins survived, and 16.5% and
13.4% with agglutinins died. The HA antibody response of the vac-
cinated birds in the present study had essentially no bearing on
the immune status of the birds. Birds that contracted the disease
had a titer range similar to those that survived. It thus appears
either that some mechanism other than circulating antibody is re-
sponsible for immunity, or that protective antibodies are not de-
tected by the usual serological methods. This idea is further sup-
ported by the failure of serum from immune birds to confer passive
protection on mice. Heddleston and Watko (12) had made a similar
observation in the chicken sera.
SUMMARY
1. All four bacterins conferred significant immunity in tur-
keys to experimental fowl cholera.
2. Heat-inactivated bacterin containing type-A P. multocida
strain gave better immunity in turkeys than formalinized bac-
terins, as indicated by higher survival and lower recovery rates
of Pasteurella organisms from the survivors.
3. No clear-cut difference in immunizing efficiencies between
oil-adjuvant and aluminum hydroxide bacterins could be estab-
lished.
4. The HA test did not clearly indicate the immune status
of the birds that were vaccinated and exposed to virulent P. multo-
cida cultures.
5. Serum of immune turkeys did not induce passive immunity
in adult mice.
REFERENCES
1. Bierer, B. W., C. L. Vickers, and H. D. Valentine. Preparation and use
of an autogenous fowl cholera bacterin. J. Am. Vet. Med. Assoc. 138, 85-86,
1961.
2. Carter, G. R. Studies on a Pasteurella multocida chicken embryo vac-
cine. I. The comparative immunizing value of broth bacterins and a chicken
embryo vaccine in mice. Am. J. Vet. Res. 11, 252-255, 1950.

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions
168 Jagmohan L. Bhasin and Ernst L. Biberstein

3. Carter, G. R. Studies on Pasteurella multocida. I. A hemagglutinal test

for the identification of serological types. Am. J. Vet. Res. 16, 481-484, 1955.
4. Chute, H. L., D. C. O'Meara, and M. Gershman. Bacterin and drugs
for the control of experimental fowl cholera. Avian Diseases 6, 7-13, 1962.
5. Engelbrecht, F. M., P. D. Byers, B. D. Stacy, C. V. Harrison, and
E. J. King. Tissue reaction to injected aluminum and alumina in the lungs and
livers of mice, rats, guinea pigs, and rabbits. J. Pathol. Bact. 77, 407-416, 1959.
6. Freund, J. The effect of paraffin oil and myco-bacteria on antibody
formation and sensitization. Am. J. Clin. Pathol. 21, 645-656, 1951.
7. Hadley, P. B. Studies on fowl cholera. IV. The reciprocal relation of
virulent and avirulent culture in active immunization. R. I. Agr. Expt. Sta.
Bull. 159, 1914.
8. Harshfield, G. S. Chapt. 11, "Fowl cholera," p. 273-286. In: "Diseases
of poultry," 4th edition, 1959, by T. E. Biester and L. H. Shwarte. Iowa State
University Press.
9. Heddleston, K. L., and W. J. Hall. Studies on pasteurellosis. II. Com-
parative efficacy of killed vaccine against fowl cholera in chickens. Avian
Diseases 2, 322-335, 1958.
10. Heddleston, K. L., and R. C. Reisinger. Studies on pasteurellosis. III.
Control of experimental fowl cholera in chickens and turkeys with an emulsi-
fied killed vaccine. Avian Diseases 4, 431-435, 1960.
11. Heddleston, K. L., and R. C. Reisinger. Studies on pasteurellosis. IV.
Killed fowl cholera vaccine adsorbed on aluminum hydroxide. Avian Diseases
4, 431-435, 1960.
12. Heddleston, K. L., and L. P. Watko. Fowl cholera: comparison of
serological and immunogenic responses of chickens and turkeys. Avian Diseases
9, 365-376, 1965.
13. Hilbert, K. F., and H. Tax. The value of chemically killed cultures for
the control of cholera in ducks. Cornell Vet. 28, 275-280, 1938.
14. Jasmin, A. M. An improved method for demonstrating bacterial cap-
sule with particular reference to Pasteurella. J. Bact. 50, 361-363, 1945.
15. Namioka, S., and M. Murata. Serological studies on Pasteurella multo-
cida. I. A simplified method for capsule typing of the organism. Cornell Vet.
51, 498-521, 1961.
16. Priestley, F. W. Experiments on immunization against Pasteurella
septica infection. J. Comp. Pathol. 49, 340-347, 1936.
17. Rebers, P. A., K. L. Heddleston, and D. J. Ganfield. Isolation and
characterization of a toxic particulate and protective antigenic fraction from
Pasteurella multocida. Fed. Proc. 24, 698, 1965.
18. Roberts, R. S. An immunological study of Pasteurella septica. J
Comp. Pathol. 57, 261-278, 1947.
19. Van Es, L., and H. M. Martin. The value of commerical vaccines and
bacterins against fowl cholera. Nebr. Agr. Expt. Sta. Res. Bull. 18, 1920.

This content downloaded from 62.122.79.81 on Mon, 23 Jun 2014 05:56:40 AM

All use subject to JSTOR Terms and Conditions