Vaccination of Chickens Through the Drinking Water with a Live, Pasteurella multocida, Vaccine1 JOHN T. RICE2, B. W. BIERER, and J. W. DICK Poultry Science Department, Clemson University, Clemson, South Carolina 29631 (Received for publication December 8, 1977)
INTRODUCTION Immunization of chickens against fowl cholera was the subject of one of the earliest investigations into the nature of the immune response. Pasteur in 1880 (cited by Heddleston, 1972) injected an attenuated strain of Pasteurella multocida into chickens and was able to produce immunity under laboratory conditions; however, attempts to use this method outside the laboratory were unsuccessful. Hadley (1914) used an avirulent strain of P. multocida to immunize mice, but no report was made of its use in poultry. With the growth of the commercial poultry industry and greater concentrations of birds, further research on the fowl cholera problem was stimulated. Immunization of poultry against fowl cholera was unreliable until a method for the preparation of an efficacious bacterin was outlined by Heddleston and Hall (1958) and Heddleston and Reisinger (1959). These bacterins were effective provided they contained the immunotypes encountered in the field. Oral immunization of chickens with a killed suspension of strain X-73 was reported by Hed-
dleston and Rebers (1968). At the same time, Bierer and Eleazer (1968), Bierer and Scott (1969), and Bierer (1969) described the use of an attenuated strain of P. multocida given through the drinking water; however, the strain used for that series of trials lost its immunogenicity through laboratory passage and never saw widespread use. Bierer and Derieux (1972) described what is now known as the Clemson University (CU) strain (formerly designated CS 148) or CU vaccine given in the drinking water to turkeys. A characteristic of this vaccine is its ability to stimulate protection against heterologous strains of P. multocida. The CU strain is the first live fowl cholera vaccine to see widespread use. It was originally produced within states for local use, but is now available nationwide as a federally licensed, lyophilized vaccine. A mutant of strain P-1059 was used by Heddleston and Rebers (1972) to immunize turkeys through the drinking water, and Maheswaran et al. (1973) reported the efficacy of another strain (M-2283) when given either endotracheally or in the drinking water. Heddleston et al. (1975) investigated the use of several P. multocida strains as immunizing agents in the drinking water. Although fowl cholera is primarily a problem in turkeys, there are geographical areas where chickens, especially broiler breeders and commercial layers, are vaccinated with commercial bacterins to prevent mortality and morbidity. In spite of this, a significant number of
1 Published with the approval of the Director of the South Carolina Agricultural Experiment Station as technical contribution no. 1509. 2 Present address: A. H. Robins Co., 624 La Vista Rd., Athens, GA 30606.
1979 Poultry Sci 58:18-22
18
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
ABSTRACT Broiler-type chickens were vaccinated orally at several ages with various dilutions of the Clemson University (CU) strain of Pasteurella multocida vaccine and then challenged orally with virulent strain P-1059 or strain X-73 of P. multocida to determine the degree of protection produced by vaccination. When vaccinated at 8 and 12 weeks of age for 2 consecutive days with a 1:13 3 dilution of the vaccine and then challenged at 17 and 19 weeks of age with strain X-73 and P-1059 P. multocida, respectively, no significant levels of protection were elicited in the vaccinates. Significant levels of protection were elicited in the vaccinates when vaccinated for 2 consecutive days at 9 weeks and at 13 weeks with 1:133 and 1:30 dilutions of the vaccine respectively, and then challenged at 16 weeks with strain X-73 and at 20 weeks with strain P-1059. Broilers vaccinated at 12 and 16 weeks for 3 consecutive days and then challenged at 20 weeks with strain X-73 showed a highly significant level of protection upon challenge when compared to the controls.
BROILERS: ORAL CHOLERA VACCINATION
MATERIALS AND METHODS
Chickens. Commercially reared broilers (Ross-Arbor Acre) were obtained at 8 weeks of age from an integrated broiler producer. None of the birds had a history of disease problems and had not been used previously for any type of research work. They were placed in concrete block pens measuring 2.7 X 3.4 m. Before each experiment the rooms were cleaned, disinfected, and 8 cm of clean shavings placed on the floor. Cultures. The Clemson University strain of P. multocida was isolated in 1970 and has been maintained by weekly passage of 10% bovine blood agar plates. Single colonies are never used in transfer procedures. The vaccine was prepared by inoculating this culture into a flask of brain-heart infusion broth and incubating it at 37 C for 16 to 18 hr. A concentration of approximately one billion viable organisms per ml was determined by standard microbiological methods. Strains X-73 and P-1059 used for challenge were obtained from K. L. Heddleston, Senior Research Bacteriologist at the National Animal Disease Center, Ames, IA. Challenge strains were grown in the same manner as the CU strain and yielded approximately the same concentration. Vaccination. A 16 to 18 hr culture of the CU strain in brain-heart infusion broth was diluted with tap water and substituted for the regular drinking water. To encourage consumption of the treated water, drinking water was withheld from the birds for a period of time before providing the vaccine-treated water. A freshly prepared flask of vaccine was used for each application. Challenge. Strain X-73 or P-1059 was ad-
ministered through the drinking water for each challenge. The culture was given as a 1:50 dilution for 3 consecutive days following 18 hr withdrawl of water. The total numbers of infected birds from the vaccinated and nonvaccinated groups at the end of the observation period were analyzed by Chi-square (Steel and Torrie, 1960). Experiment 1. One hundred and sixty chickens were randomly divided into 8 pens. Half of the pens were vaccinated for 2 consecutive days at both 8 and 12 weeks of age. Thirty milliliters of the vaccine culture were diluted 1:133 by the addition of 3970 ml of tap water. The diluted (1:133) vaccine contained approximately 7.5 x 10 6 bacilli/ml. Drinking water was withdrawn from the birds 2 hours prior to each oral vaccination to encourage consumption. Sixty birds from each treatment group were challenged at 17 weeks of age with strain X-73. Twenty birds from each treatment were challenged at 19 weeks of age with strain P1059. Mortality and morbidity were noted daily for 8 days postchallenge. The livers from a representative number of birds that died were cultured in order to establish infection with the challenge organism. Experiment 2. Twenty-two broiler chickens were placed in each of eight pens. Four of these pens were vaccinated at 9 weeks of age with a 1:133 dilution of the CU vaccine as described in Experiment 1. At 13 weeks of age they were revaccinated, but with a 1:50 dilution for 3 consecutive days following 18 hr withdrawal of drinking water prior to each administration. The withdrawal period extended from 1600 hr in the afternoon until 1000 hr the following morning. The 1:50 dilution contained approximately 20 million viable organisms per ml. At 16 weeks of age 48 vaccinated and 48 nonvaccinated chickens were challenged orally with a virulent X-73 strain of P. multocida. At 20 weeks of age 40 birds from each group were challenged with strain P-1059. Mortality and morbidity were recorded for 14 days after each challenge. Experiment 3. Sixteen eight-week-old broilers were placed in each of eight pens. Four pens of birds were vaccinated at 12 and 16 weeks with a 1:50 dilution of the CU vaccine, given for 3 consecutive days following 18 hours withdrawal of drinking water. All birds were challenged at 20 weeks of age through the drinking water with strain X-73 of P. multocida and observed for 2 weeks.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
producers have had difficulty obtaining a protective level of immunity in chickens. Bacterins which have been used often do not protect against serotypes encountered in the field; hence, producers who have flocks with fowl cholera problems want to use a vaccine that will protect against heterologous strains of P. multocida. The fact that fowl cholera today is more prevalent in turkeys than in chickens probably accounts for the little work that has been done with chickens. Consequently, this study was undertaken to determine whether the Clemson University vaccine given in the drinking water of chickens would stimulate immunity to an experimental infection with P. multocida.
19
20
RICE ET AL. RESULTS AND DISCUSSION
Jl d« Z
U
rt rt .O Xi IA m O N Tj- T t »H i-H
J-. . «
O T3
II -a
z >z
a " = =
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
The use of the CU vaccine on the 232 chickens vaccinated in the three experiments described produced no mortality or morbidity in the time period between vaccination and challenge. Since the CU vaccine strain reacts with type 3 (P-1059) and type 4 (P-1662) antisera when subjected to a gel diffusion test described by Heddleston (1972), the X-73 (type 1) challenges indicated in these experiments were heterologous challenges (Derieux, 1974). Experiment 1. Mortality and morbidity following challenge with P. multocida strain X-73 are shown in Table 1. The experiment was terminated at 8 days postchallenge. Statistical analysis of the total numbers of infected birds in the two treatment groups indicated that no significant level of immunity was obtained by the administration of 1:133 dilution of the CU vaccine at 8 weeks and repeated at 12 weeks of age. Mortality and morbidity following challenge of the remaining birds with strain P-1059 are also shown in Table 1. No difference in resistance was seen between the vaccinated and nonvaccinated groups. Thus, under the conditions of this experiment, a 1:133 dilution of the CU vaccine, which was shown by Bierer and Derieux (1972) to induce immunity in turkeys, failed to provide protection in chickens against the virulent strains used. Experiment 2, No detrimental effects from the vaccination or water withdrawal were noted in any birds. Table 2 shows the mortality and morbidity in experiment 2 during 2 weeks after challenge with the virulent X-73 strain through the drinking water. The differences in the total numbers of infected birds are highly significant (P«.01). The results of the P-1059 challenge given 7 weeks after vaccination are also shown in Table 2. Analysis of these data showed that difference in the total numbers of birds infected with this strain was also highly significant (P«.01). Experiment 3. All birds in this experiment were challenged with the X-73 strain of P. multocida 4 weeks after the second vaccination period. At the end of the 14 day observation period there were fewer birds infected in the vaccinated group than in the nonvaccinated (see Table 3). This difference was significant (P«.01). The results of trials 2 and 3 indicate that the use of a 1:50 dilution of the CU vaccine, repeated after a 4 week interval, can provide a significant degree of immunity in chickens
1 0 0 1
2
3 6 0 2
3
3 7 0 1
4 1 6 3 7
5 2 3 1 3
6 2 3 2 4
7
8 0 1 1 0
9 1 0 0 0
Mortality - days post-challenge
0 0 0 2
10 1 1 1 0
11
3 9
2
3
5 13
1 17
4
5 1 2 1 4
6
7 1 2
1 2
8
9 1 2
All birds were challenged 4 weeks post-vaccination.
1 2
10
in orally vaccinated and nonvaccinated broilers strain X-73, P. multocida. Experiment 3
Mortality - days post-challenge
and morbidity
ab ' Values followed by different letters are significantly different by pairwise Chi square analysis (P
INTRODUCTION Immunization of chickens against fowl cholera was the subject of one of the earliest investigations into the nature of the immune response. Pasteur in 1880 (cited by Heddleston, 1972) injected an attenuated strain of Pasteurella multocida into chickens and was able to produce immunity under laboratory conditions; however, attempts to use this method outside the laboratory were unsuccessful. Hadley (1914) used an avirulent strain of P. multocida to immunize mice, but no report was made of its use in poultry. With the growth of the commercial poultry industry and greater concentrations of birds, further research on the fowl cholera problem was stimulated. Immunization of poultry against fowl cholera was unreliable until a method for the preparation of an efficacious bacterin was outlined by Heddleston and Hall (1958) and Heddleston and Reisinger (1959). These bacterins were effective provided they contained the immunotypes encountered in the field. Oral immunization of chickens with a killed suspension of strain X-73 was reported by Hed-
dleston and Rebers (1968). At the same time, Bierer and Eleazer (1968), Bierer and Scott (1969), and Bierer (1969) described the use of an attenuated strain of P. multocida given through the drinking water; however, the strain used for that series of trials lost its immunogenicity through laboratory passage and never saw widespread use. Bierer and Derieux (1972) described what is now known as the Clemson University (CU) strain (formerly designated CS 148) or CU vaccine given in the drinking water to turkeys. A characteristic of this vaccine is its ability to stimulate protection against heterologous strains of P. multocida. The CU strain is the first live fowl cholera vaccine to see widespread use. It was originally produced within states for local use, but is now available nationwide as a federally licensed, lyophilized vaccine. A mutant of strain P-1059 was used by Heddleston and Rebers (1972) to immunize turkeys through the drinking water, and Maheswaran et al. (1973) reported the efficacy of another strain (M-2283) when given either endotracheally or in the drinking water. Heddleston et al. (1975) investigated the use of several P. multocida strains as immunizing agents in the drinking water. Although fowl cholera is primarily a problem in turkeys, there are geographical areas where chickens, especially broiler breeders and commercial layers, are vaccinated with commercial bacterins to prevent mortality and morbidity. In spite of this, a significant number of
1 Published with the approval of the Director of the South Carolina Agricultural Experiment Station as technical contribution no. 1509. 2 Present address: A. H. Robins Co., 624 La Vista Rd., Athens, GA 30606.
1979 Poultry Sci 58:18-22
18
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
ABSTRACT Broiler-type chickens were vaccinated orally at several ages with various dilutions of the Clemson University (CU) strain of Pasteurella multocida vaccine and then challenged orally with virulent strain P-1059 or strain X-73 of P. multocida to determine the degree of protection produced by vaccination. When vaccinated at 8 and 12 weeks of age for 2 consecutive days with a 1:13 3 dilution of the vaccine and then challenged at 17 and 19 weeks of age with strain X-73 and P-1059 P. multocida, respectively, no significant levels of protection were elicited in the vaccinates. Significant levels of protection were elicited in the vaccinates when vaccinated for 2 consecutive days at 9 weeks and at 13 weeks with 1:133 and 1:30 dilutions of the vaccine respectively, and then challenged at 16 weeks with strain X-73 and at 20 weeks with strain P-1059. Broilers vaccinated at 12 and 16 weeks for 3 consecutive days and then challenged at 20 weeks with strain X-73 showed a highly significant level of protection upon challenge when compared to the controls.
BROILERS: ORAL CHOLERA VACCINATION
MATERIALS AND METHODS
Chickens. Commercially reared broilers (Ross-Arbor Acre) were obtained at 8 weeks of age from an integrated broiler producer. None of the birds had a history of disease problems and had not been used previously for any type of research work. They were placed in concrete block pens measuring 2.7 X 3.4 m. Before each experiment the rooms were cleaned, disinfected, and 8 cm of clean shavings placed on the floor. Cultures. The Clemson University strain of P. multocida was isolated in 1970 and has been maintained by weekly passage of 10% bovine blood agar plates. Single colonies are never used in transfer procedures. The vaccine was prepared by inoculating this culture into a flask of brain-heart infusion broth and incubating it at 37 C for 16 to 18 hr. A concentration of approximately one billion viable organisms per ml was determined by standard microbiological methods. Strains X-73 and P-1059 used for challenge were obtained from K. L. Heddleston, Senior Research Bacteriologist at the National Animal Disease Center, Ames, IA. Challenge strains were grown in the same manner as the CU strain and yielded approximately the same concentration. Vaccination. A 16 to 18 hr culture of the CU strain in brain-heart infusion broth was diluted with tap water and substituted for the regular drinking water. To encourage consumption of the treated water, drinking water was withheld from the birds for a period of time before providing the vaccine-treated water. A freshly prepared flask of vaccine was used for each application. Challenge. Strain X-73 or P-1059 was ad-
ministered through the drinking water for each challenge. The culture was given as a 1:50 dilution for 3 consecutive days following 18 hr withdrawl of water. The total numbers of infected birds from the vaccinated and nonvaccinated groups at the end of the observation period were analyzed by Chi-square (Steel and Torrie, 1960). Experiment 1. One hundred and sixty chickens were randomly divided into 8 pens. Half of the pens were vaccinated for 2 consecutive days at both 8 and 12 weeks of age. Thirty milliliters of the vaccine culture were diluted 1:133 by the addition of 3970 ml of tap water. The diluted (1:133) vaccine contained approximately 7.5 x 10 6 bacilli/ml. Drinking water was withdrawn from the birds 2 hours prior to each oral vaccination to encourage consumption. Sixty birds from each treatment group were challenged at 17 weeks of age with strain X-73. Twenty birds from each treatment were challenged at 19 weeks of age with strain P1059. Mortality and morbidity were noted daily for 8 days postchallenge. The livers from a representative number of birds that died were cultured in order to establish infection with the challenge organism. Experiment 2. Twenty-two broiler chickens were placed in each of eight pens. Four of these pens were vaccinated at 9 weeks of age with a 1:133 dilution of the CU vaccine as described in Experiment 1. At 13 weeks of age they were revaccinated, but with a 1:50 dilution for 3 consecutive days following 18 hr withdrawal of drinking water prior to each administration. The withdrawal period extended from 1600 hr in the afternoon until 1000 hr the following morning. The 1:50 dilution contained approximately 20 million viable organisms per ml. At 16 weeks of age 48 vaccinated and 48 nonvaccinated chickens were challenged orally with a virulent X-73 strain of P. multocida. At 20 weeks of age 40 birds from each group were challenged with strain P-1059. Mortality and morbidity were recorded for 14 days after each challenge. Experiment 3. Sixteen eight-week-old broilers were placed in each of eight pens. Four pens of birds were vaccinated at 12 and 16 weeks with a 1:50 dilution of the CU vaccine, given for 3 consecutive days following 18 hours withdrawal of drinking water. All birds were challenged at 20 weeks of age through the drinking water with strain X-73 of P. multocida and observed for 2 weeks.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
producers have had difficulty obtaining a protective level of immunity in chickens. Bacterins which have been used often do not protect against serotypes encountered in the field; hence, producers who have flocks with fowl cholera problems want to use a vaccine that will protect against heterologous strains of P. multocida. The fact that fowl cholera today is more prevalent in turkeys than in chickens probably accounts for the little work that has been done with chickens. Consequently, this study was undertaken to determine whether the Clemson University vaccine given in the drinking water of chickens would stimulate immunity to an experimental infection with P. multocida.
19
20
RICE ET AL. RESULTS AND DISCUSSION
Jl d« Z
U
rt rt .O Xi IA m O N Tj- T t »H i-H
J-. . «
O T3
II -a
z >z
a " = =
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
The use of the CU vaccine on the 232 chickens vaccinated in the three experiments described produced no mortality or morbidity in the time period between vaccination and challenge. Since the CU vaccine strain reacts with type 3 (P-1059) and type 4 (P-1662) antisera when subjected to a gel diffusion test described by Heddleston (1972), the X-73 (type 1) challenges indicated in these experiments were heterologous challenges (Derieux, 1974). Experiment 1. Mortality and morbidity following challenge with P. multocida strain X-73 are shown in Table 1. The experiment was terminated at 8 days postchallenge. Statistical analysis of the total numbers of infected birds in the two treatment groups indicated that no significant level of immunity was obtained by the administration of 1:133 dilution of the CU vaccine at 8 weeks and repeated at 12 weeks of age. Mortality and morbidity following challenge of the remaining birds with strain P-1059 are also shown in Table 1. No difference in resistance was seen between the vaccinated and nonvaccinated groups. Thus, under the conditions of this experiment, a 1:133 dilution of the CU vaccine, which was shown by Bierer and Derieux (1972) to induce immunity in turkeys, failed to provide protection in chickens against the virulent strains used. Experiment 2, No detrimental effects from the vaccination or water withdrawal were noted in any birds. Table 2 shows the mortality and morbidity in experiment 2 during 2 weeks after challenge with the virulent X-73 strain through the drinking water. The differences in the total numbers of infected birds are highly significant (P«.01). The results of the P-1059 challenge given 7 weeks after vaccination are also shown in Table 2. Analysis of these data showed that difference in the total numbers of birds infected with this strain was also highly significant (P«.01). Experiment 3. All birds in this experiment were challenged with the X-73 strain of P. multocida 4 weeks after the second vaccination period. At the end of the 14 day observation period there were fewer birds infected in the vaccinated group than in the nonvaccinated (see Table 3). This difference was significant (P«.01). The results of trials 2 and 3 indicate that the use of a 1:50 dilution of the CU vaccine, repeated after a 4 week interval, can provide a significant degree of immunity in chickens
1 0 0 1
2
3 6 0 2
3
3 7 0 1
4 1 6 3 7
5 2 3 1 3
6 2 3 2 4
7
8 0 1 1 0
9 1 0 0 0
Mortality - days post-challenge
0 0 0 2
10 1 1 1 0
11
3 9
2
3
5 13
1 17
4
5 1 2 1 4
6
7 1 2
1 2
8
9 1 2
All birds were challenged 4 weeks post-vaccination.
1 2
10
in orally vaccinated and nonvaccinated broilers strain X-73, P. multocida. Experiment 3
Mortality - days post-challenge
and morbidity
ab ' Values followed by different letters are significantly different by pairwise Chi square analysis (P
