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Laboratory Studies on Erysipelas
Laboratory Studies on Erysipelas 4. DURATION OF IMMUNITY IN TURKEYS VACCINATED WITH AN ADSORBED BACTERIN M. S. COOPER, G. R. PERSONEUS AND R. C. PERCIVAL Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York, and South St. Joseph, Missouri (Received for publication August 13, 1956)
This report presents results of further studies indicating the duration of immunity conferred on turkeys by inoculation with various dosages of the adsorbed bacterin against intravenous challenge. MATERIALS AND METHODS
Bacterins. The erysipelas bacterins were prepared from chemically killed, whole broth cultures of E. rhusiopathiae, precipitated with aluminum hydroxide, concentrated, and potency-tested in mice. An experimental lot was used in Experiment 1, and a commercial lot in Experiment 2. To permit comparison of the test results of the two experiments, the potency of both bacterins was compared.
Threefold dilutions of the bacterins were prepared in physiological saline. Each dilution was injected subcutaneously into groups of 16 white mice (Table 1). Two weeks later, all groups were challenged subcutaneously with 0.5 ml. of a 10~5 dilution (about 1,000 MLD) of an 18-hour broth culture of E. rhusiopathiae, with results summarized in Table 1. A statistical analysis (Litchfield and Wilcoxon, 1949) of the mouse potency results indicates that the 95% confidence limits of the PD50 of each bacterin are as follows: Experimental bacterin Commercial bacterin
0.00204 ml.-0.0060 ml. 0.00172 ml.-0.00488 ml-
The difference in potency of the two bacterins was found to be not significant, since the results overlap almost completely. Challenge Cultures. Virulent cultures of E. rhusiopathiae were grown for 18 hours at 37°C. in yeast bile broth containing 5% horse serum. Experiment 1. Seventy-two Beltsville White poults, 6 to 8 weeks of age, were placed by random selection in four pens of 15 each for vaccination, and one pen of
* Duovax R Erysipelas Bacterin.
266
TABLE 1.—Potency tests of bacterins in mice Final dilution injected
Survivors/ Total challenged
Experimental
1:100 1:300 1:900
14/16 9/16 3/16
Commercial
1:150 1:450 1:1,350
10/16 7/16 0/16 0/16
Bacterin
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
' I VHE development of an adsorbed -*- bacterin* for immunization against infection by Erysipelothrix rkusiopaihiae was described by Cooper, Personeus and Choman (1954). In this study, it was also shown that turkeys could be successfully immunized against a laboratory challenge. Two hundred and fifty-four 4- to 6-weeksold poults were injected intramuscularly with 1 ml. of bacterin, and 3 weeks later, together with 62 nonvaccinated controls, were challenged intramuscularly with 1 ml., or intravenously with 0.5 ml. of erysipelas culture. All of 183 vaccinated poults, challenged intramuscularly, survived challenge which killed 88% of the 54 controls; 95.8% of 71 challenged intravenously survived, while all 8 controls died.
267
ERYSIPELAS VACCINATION OF TURKEYS
RESULTS
TABLE 3.—Results of challenge of turkeys vaccinated with commercial adsorbed erysipelas bacterin Time of challenge postvaccination i—weeks
Immunizing dose 4 ml. 2 ml. 1ml. 0.5 ml. Nonvaccinated controls
6
9i
12
15
15/15* 15/15 11/15 11/15
15/15 13/15 11/15 5/15
8/15 5/15 2/15 0/15
5/14 4/14 2/13 0/15
2/15
1/15
0/15
0/13
* Survivors/total challenged.
than 1:32 in turkeys against an intravenous challenge. Experiment 2. Challenge results (Table 3) indicated a longer duration of immunity with thelarger doses of bacterin. When vaccinated with 4 ml., all challenged birds survived through 9 | weeks, 8 of 18 through 12 weeks, and 5 of 14 through 15 weeks, compared with death of 13 of 15 controls at 6 weeks, 14 of 15 at 9f weeks, and all challenged at 12 and 15 weeks. Protection afforded by the 2 ml. dose was only slightly less. Results of statistical analysis, presented in Table 4 and Figure 1, further substantiate this observation. These results are expressed in terms of DI 50 (duration of immunity), or the length of time after vaccination when 50% of the animals survived challenge.
Experiment 1. Deaths usually occurred between the third and fifth postchallenge days. Challenge results, summarized in Table 2, indicate that the 50% protective dose of experimental bacterin was less
In Experiment 1, it was established that 1:32 dilution of an experimental erysipelas bacterin protected more than 50% of turkeys against a severe laboratory
TABLE 2.—Results of challenge of turkeys vaccinated with experimental adsorbed erysipelas bacterin
TABLE 4.—Statistical analysis of challenge results
Final concentration of vaccine injected 1:2 (0.5 ml.) 1:5 (0.2 ml.) 1:12.5 (0.08 ml.) 1:32 (0.032 ml.) Nonvaccinated controls
Survivors/total challenged 15/15 13/15 14/15 12/15 0/12
DISCUSSION
Dose 4 ml. 2 ml. lml. 0.5 ml. :
DI 60 * 12.5 11.9 9.3 7.9
weeks weeks weeks weeks
95% confidence limits of DI6o 9.5-16.4 10.5-13.6 7.6-11.4 6 . 4 - 9.8
weeks weeks weeks weeks
50% endpoint in duration of immunity.
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
12 un vaccina ted controls. Birds were injected intramuscularly in the thigh with various dilutions of experimental bacterin prepared in physiological saline. The final concentrations contained in the 0.5-ml. amounts injected were 1:2, 1:5, 1:12.5, and 1:32. Two weeks later, both vaccinated and control birds were challenged intravenously with 0.5 ml. of a virulent culture of E. rhusiopathiae. Turkeys were observed for two weeks postchallenge. Experiment 2. One hundred ninetyeight White Holland and 100 Broad Breasted Bronze turkeys, about 10 weeks of age, were divided at random into four groups of 60 each and one group of 58, each group containing proportionate numbers of bronze and white birds. Those in the four groups of 60 each were vaccinated intramuscularly in the thigh with 0.5, 1.0, 2.0, or 4.0 ml. of bacterin, respectively, and the remaining 58 were held unvaccinated as controls. Challenge was carried out by injection into the wing vein of 0.5 ml. of a 10~6 dilution of E. rhusiopathiae culture. Fifteen birds from each of the vaccinated and control groups were challenged at 6, 9§, 12, and 15 weeks, respectively, again with proportionate numbers of bronze and white birds in each group.
268
M. S. COOPER, G. R. PERSONEUS AND R. C. PERCIVAL
FIG. 1. Percentage survival of turkeys after immunization with different doses of bacterin.
challenge given two weeks after vaccination. In Experiment 2, the higher doses of vaccine, 2 and 4 ml., gave longer protection than the lowest dose of 0.5 ml. The minimum dose used in the latter studywas at least 15 times the concentration of the minimum effective dose in the short term study. These results suggest that the dose concentration and the greater persistence of the depot due to a greater volume of bacterin (and adjuvant) used are important factors in determining the duration of the immune response even where the lowest dosage used is many times the minimum effective concentration as determined at short term challenge. The intravenous challenge used in these studies is usually considered extremely severe (Dickinson et al., 1953; Jerstad and Johns, 1954b), and results obtained cannot be used to establish an absolute estimate of the duration of immunity under natural field exposure. This bacterin has been rather widely field tested in turkey flocks where erysipelas had been a problem in years past (Adler and Nilson, 1952; Adler and Spencer, 1952; Dickinson et al., 1953; Jerstad and Johns, 1954a, b). In these trials, conducted over a two-year period, 62,144 birds from 53 flocks were vaccinated, and 35,251 were left unvac-
SUMMARY
Although the minimum effective dose of an experimental erysipelas bacterin was less than 0.032 ml. in turkeys, 2 and 4 ml. of a similar commercial bacterin produced significantly longer immunity than 0.5 ml. ACKNOWLEDGMENT
The authors wish to express their appreciation to Dr. F. Wilcoxon and Mr. C. Dunnett for the statistical analyses, to Mr. R. Babcock, for help in performing the turkey studies, and to Mrs. D. Budd for assistance in preparation of the manuscript. REFERENCES Adler, H. E., and M. A. Nilson, 1952. Immunization of turkeys against swine erysipelas with several types of bacterins. Canad. J. Comp. Med. Vet. Sci. 16:390-393. Adler, H. E., and G. R. Spencer, 1952. Immunization of turkeys and pigs with an erysipelas bacterin. Cornell Vet. 42: 238-246. Cooper, M. S., G. R. Personeus and R. B. Choman, 1954. Laboratory studies on the vaccination of mice and turkeys with an Erysipelothrix rkusiopathiae vaccine. Canad. J. Comp. Med. 18: 83-92. Dickinson, E. M., A. C. Jerstad, H. E. Adler, M. S.
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
Dose of Vaccine, ml.
cinated as controls. All ages of birds were represented, from 6-weeks-old poults to mature breeders. In general, a single intramuscular injection of 2 ml. of the bacterin increased resistance to erysipelas during the subsequent season when natural outbreaks occurred in nonvaccinated controls. From 39 flocks, 434 vaccinated and 377 nonvaccinated turkeys were challenged 5 to 40 weeks postvaccination by the intravenous injection of various amounts of a broth culture of E. rhusiopathiae. It was found that birds vaccinated at 6 to 8 weeks of age withstood this severe challenge better than those vaccinated at a later age, and that, especially in older birds, a second immunizing injection increased immunity,
ERYSIPELAS VACCINATION OF TURKEYS Cooper, W. E. Babcock, E. E. Johns and C. A. Bottorff, 1953. The use of an Erysipelothrix rhusiopathiae bacterin for the control of erysipelas in turkeys. Proc. Book Am. Vet. Med. Assoc: 370-375. Jerstad, A. C , and E. E. Johns, 1954a. Performance of a bacterin in the control of erysipelas in tur-
269
keys. Proc. Book Am. Vet. Med. Assoc: 333-337. Jerstad, A. C , and E. E. Johns, 1954b. Field trials of a bacterin for the control of erysipelas in turkeys. J. Am. Vet. Med. Assoc. 125: 288-291. Litchfield, J. T., and F. A. Wilcoxon, 1949. A simplified method of evaluating dose-effect experiment. J. Pharmacol. Exp. Therap. 96:99-113.
ENSEL C. STUTTS, W. E. BRILES AND H. 0 . KUNKEL Departments of Biochemistry and Nutrition and of Poultry Science, Texas Agricultural Experiment Station, College Station, Texas (Received for publication August 24, 1956)
A
RE VIE W of the existent literature reveals very little information concerning the metabolic mechanisms by which genes are able to control the productivity of birds or animals. One approach to the problem of gene action is a study of biochemical variation and individuality, for it is difficult to conceive of any biological diversity, including productivity, which does not reside in the biochemistry of the individual. To be of greatest value in experimentally relating heredity, metabolism, and production, a particular biochemical difference should be largely under genetic control so that it is relatively stable to environmental influences. An example of such a biochemical individuality is the pattern of glutathione metabolism in the erythrocytes, where there are distinct individual and line differences in inbred chickens (Stutts et al., 1956a) and individual and breed differences in immature beef cattle (Kunkel et al., 1954). Subsequent investigation has resulted in preliminary evidence of a significant negative correlation between the blood reduced glutathione level and egg production in the inbred chickens (Stutts et al., 1956b)
and a significant curvilinear correlation between erythrocyte reduced glutathione and rate of gain in certain breeds of cattle (Kunkel, 1956). The investigation of the extent of the genetic control of biochemical patterns known to be affected by environmental forces is also of interest. The plasma or serum alkaline phosphatase in rats can be reduced by fasting 24 hours (Jackson, 1952) and this reduction is ameliorated by dietary protein and fat, and probably by supplemental choline (Tuba and Madsen, 1952). The serum alkaline phosphatase is increased in vitamin D deficient chicks (Motzok, 1950), decreased in manganese deficient chicks (Wiese et al., 1939), and increased with the administration of growth hormone to rats (Li el al., 1947). Breed differences as well as the considerable individual variation within each of three breeds of chickens were noted by Auchinachie and Emslie (1934). Common (1934, 1936) reported that the average phosphatase activity of sexually immature pullets is comparable with the average level in young cockerels. There was no significant difference between the phosphatase values of laying and those of
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
Plasma Alkaline Phosphatase Activity in Mature Inbred Chickens
This report presents results of further studies indicating the duration of immunity conferred on turkeys by inoculation with various dosages of the adsorbed bacterin against intravenous challenge. MATERIALS AND METHODS
Bacterins. The erysipelas bacterins were prepared from chemically killed, whole broth cultures of E. rhusiopathiae, precipitated with aluminum hydroxide, concentrated, and potency-tested in mice. An experimental lot was used in Experiment 1, and a commercial lot in Experiment 2. To permit comparison of the test results of the two experiments, the potency of both bacterins was compared.
Threefold dilutions of the bacterins were prepared in physiological saline. Each dilution was injected subcutaneously into groups of 16 white mice (Table 1). Two weeks later, all groups were challenged subcutaneously with 0.5 ml. of a 10~5 dilution (about 1,000 MLD) of an 18-hour broth culture of E. rhusiopathiae, with results summarized in Table 1. A statistical analysis (Litchfield and Wilcoxon, 1949) of the mouse potency results indicates that the 95% confidence limits of the PD50 of each bacterin are as follows: Experimental bacterin Commercial bacterin
0.00204 ml.-0.0060 ml. 0.00172 ml.-0.00488 ml-
The difference in potency of the two bacterins was found to be not significant, since the results overlap almost completely. Challenge Cultures. Virulent cultures of E. rhusiopathiae were grown for 18 hours at 37°C. in yeast bile broth containing 5% horse serum. Experiment 1. Seventy-two Beltsville White poults, 6 to 8 weeks of age, were placed by random selection in four pens of 15 each for vaccination, and one pen of
* Duovax R Erysipelas Bacterin.
266
TABLE 1.—Potency tests of bacterins in mice Final dilution injected
Survivors/ Total challenged
Experimental
1:100 1:300 1:900
14/16 9/16 3/16
Commercial
1:150 1:450 1:1,350
10/16 7/16 0/16 0/16
Bacterin
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
' I VHE development of an adsorbed -*- bacterin* for immunization against infection by Erysipelothrix rkusiopaihiae was described by Cooper, Personeus and Choman (1954). In this study, it was also shown that turkeys could be successfully immunized against a laboratory challenge. Two hundred and fifty-four 4- to 6-weeksold poults were injected intramuscularly with 1 ml. of bacterin, and 3 weeks later, together with 62 nonvaccinated controls, were challenged intramuscularly with 1 ml., or intravenously with 0.5 ml. of erysipelas culture. All of 183 vaccinated poults, challenged intramuscularly, survived challenge which killed 88% of the 54 controls; 95.8% of 71 challenged intravenously survived, while all 8 controls died.
267
ERYSIPELAS VACCINATION OF TURKEYS
RESULTS
TABLE 3.—Results of challenge of turkeys vaccinated with commercial adsorbed erysipelas bacterin Time of challenge postvaccination i—weeks
Immunizing dose 4 ml. 2 ml. 1ml. 0.5 ml. Nonvaccinated controls
6
9i
12
15
15/15* 15/15 11/15 11/15
15/15 13/15 11/15 5/15
8/15 5/15 2/15 0/15
5/14 4/14 2/13 0/15
2/15
1/15
0/15
0/13
* Survivors/total challenged.
than 1:32 in turkeys against an intravenous challenge. Experiment 2. Challenge results (Table 3) indicated a longer duration of immunity with thelarger doses of bacterin. When vaccinated with 4 ml., all challenged birds survived through 9 | weeks, 8 of 18 through 12 weeks, and 5 of 14 through 15 weeks, compared with death of 13 of 15 controls at 6 weeks, 14 of 15 at 9f weeks, and all challenged at 12 and 15 weeks. Protection afforded by the 2 ml. dose was only slightly less. Results of statistical analysis, presented in Table 4 and Figure 1, further substantiate this observation. These results are expressed in terms of DI 50 (duration of immunity), or the length of time after vaccination when 50% of the animals survived challenge.
Experiment 1. Deaths usually occurred between the third and fifth postchallenge days. Challenge results, summarized in Table 2, indicate that the 50% protective dose of experimental bacterin was less
In Experiment 1, it was established that 1:32 dilution of an experimental erysipelas bacterin protected more than 50% of turkeys against a severe laboratory
TABLE 2.—Results of challenge of turkeys vaccinated with experimental adsorbed erysipelas bacterin
TABLE 4.—Statistical analysis of challenge results
Final concentration of vaccine injected 1:2 (0.5 ml.) 1:5 (0.2 ml.) 1:12.5 (0.08 ml.) 1:32 (0.032 ml.) Nonvaccinated controls
Survivors/total challenged 15/15 13/15 14/15 12/15 0/12
DISCUSSION
Dose 4 ml. 2 ml. lml. 0.5 ml. :
DI 60 * 12.5 11.9 9.3 7.9
weeks weeks weeks weeks
95% confidence limits of DI6o 9.5-16.4 10.5-13.6 7.6-11.4 6 . 4 - 9.8
weeks weeks weeks weeks
50% endpoint in duration of immunity.
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
12 un vaccina ted controls. Birds were injected intramuscularly in the thigh with various dilutions of experimental bacterin prepared in physiological saline. The final concentrations contained in the 0.5-ml. amounts injected were 1:2, 1:5, 1:12.5, and 1:32. Two weeks later, both vaccinated and control birds were challenged intravenously with 0.5 ml. of a virulent culture of E. rhusiopathiae. Turkeys were observed for two weeks postchallenge. Experiment 2. One hundred ninetyeight White Holland and 100 Broad Breasted Bronze turkeys, about 10 weeks of age, were divided at random into four groups of 60 each and one group of 58, each group containing proportionate numbers of bronze and white birds. Those in the four groups of 60 each were vaccinated intramuscularly in the thigh with 0.5, 1.0, 2.0, or 4.0 ml. of bacterin, respectively, and the remaining 58 were held unvaccinated as controls. Challenge was carried out by injection into the wing vein of 0.5 ml. of a 10~6 dilution of E. rhusiopathiae culture. Fifteen birds from each of the vaccinated and control groups were challenged at 6, 9§, 12, and 15 weeks, respectively, again with proportionate numbers of bronze and white birds in each group.
268
M. S. COOPER, G. R. PERSONEUS AND R. C. PERCIVAL
FIG. 1. Percentage survival of turkeys after immunization with different doses of bacterin.
challenge given two weeks after vaccination. In Experiment 2, the higher doses of vaccine, 2 and 4 ml., gave longer protection than the lowest dose of 0.5 ml. The minimum dose used in the latter studywas at least 15 times the concentration of the minimum effective dose in the short term study. These results suggest that the dose concentration and the greater persistence of the depot due to a greater volume of bacterin (and adjuvant) used are important factors in determining the duration of the immune response even where the lowest dosage used is many times the minimum effective concentration as determined at short term challenge. The intravenous challenge used in these studies is usually considered extremely severe (Dickinson et al., 1953; Jerstad and Johns, 1954b), and results obtained cannot be used to establish an absolute estimate of the duration of immunity under natural field exposure. This bacterin has been rather widely field tested in turkey flocks where erysipelas had been a problem in years past (Adler and Nilson, 1952; Adler and Spencer, 1952; Dickinson et al., 1953; Jerstad and Johns, 1954a, b). In these trials, conducted over a two-year period, 62,144 birds from 53 flocks were vaccinated, and 35,251 were left unvac-
SUMMARY
Although the minimum effective dose of an experimental erysipelas bacterin was less than 0.032 ml. in turkeys, 2 and 4 ml. of a similar commercial bacterin produced significantly longer immunity than 0.5 ml. ACKNOWLEDGMENT
The authors wish to express their appreciation to Dr. F. Wilcoxon and Mr. C. Dunnett for the statistical analyses, to Mr. R. Babcock, for help in performing the turkey studies, and to Mrs. D. Budd for assistance in preparation of the manuscript. REFERENCES Adler, H. E., and M. A. Nilson, 1952. Immunization of turkeys against swine erysipelas with several types of bacterins. Canad. J. Comp. Med. Vet. Sci. 16:390-393. Adler, H. E., and G. R. Spencer, 1952. Immunization of turkeys and pigs with an erysipelas bacterin. Cornell Vet. 42: 238-246. Cooper, M. S., G. R. Personeus and R. B. Choman, 1954. Laboratory studies on the vaccination of mice and turkeys with an Erysipelothrix rkusiopathiae vaccine. Canad. J. Comp. Med. 18: 83-92. Dickinson, E. M., A. C. Jerstad, H. E. Adler, M. S.
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
Dose of Vaccine, ml.
cinated as controls. All ages of birds were represented, from 6-weeks-old poults to mature breeders. In general, a single intramuscular injection of 2 ml. of the bacterin increased resistance to erysipelas during the subsequent season when natural outbreaks occurred in nonvaccinated controls. From 39 flocks, 434 vaccinated and 377 nonvaccinated turkeys were challenged 5 to 40 weeks postvaccination by the intravenous injection of various amounts of a broth culture of E. rhusiopathiae. It was found that birds vaccinated at 6 to 8 weeks of age withstood this severe challenge better than those vaccinated at a later age, and that, especially in older birds, a second immunizing injection increased immunity,
ERYSIPELAS VACCINATION OF TURKEYS Cooper, W. E. Babcock, E. E. Johns and C. A. Bottorff, 1953. The use of an Erysipelothrix rhusiopathiae bacterin for the control of erysipelas in turkeys. Proc. Book Am. Vet. Med. Assoc: 370-375. Jerstad, A. C , and E. E. Johns, 1954a. Performance of a bacterin in the control of erysipelas in tur-
269
keys. Proc. Book Am. Vet. Med. Assoc: 333-337. Jerstad, A. C , and E. E. Johns, 1954b. Field trials of a bacterin for the control of erysipelas in turkeys. J. Am. Vet. Med. Assoc. 125: 288-291. Litchfield, J. T., and F. A. Wilcoxon, 1949. A simplified method of evaluating dose-effect experiment. J. Pharmacol. Exp. Therap. 96:99-113.
ENSEL C. STUTTS, W. E. BRILES AND H. 0 . KUNKEL Departments of Biochemistry and Nutrition and of Poultry Science, Texas Agricultural Experiment Station, College Station, Texas (Received for publication August 24, 1956)
A
RE VIE W of the existent literature reveals very little information concerning the metabolic mechanisms by which genes are able to control the productivity of birds or animals. One approach to the problem of gene action is a study of biochemical variation and individuality, for it is difficult to conceive of any biological diversity, including productivity, which does not reside in the biochemistry of the individual. To be of greatest value in experimentally relating heredity, metabolism, and production, a particular biochemical difference should be largely under genetic control so that it is relatively stable to environmental influences. An example of such a biochemical individuality is the pattern of glutathione metabolism in the erythrocytes, where there are distinct individual and line differences in inbred chickens (Stutts et al., 1956a) and individual and breed differences in immature beef cattle (Kunkel et al., 1954). Subsequent investigation has resulted in preliminary evidence of a significant negative correlation between the blood reduced glutathione level and egg production in the inbred chickens (Stutts et al., 1956b)
and a significant curvilinear correlation between erythrocyte reduced glutathione and rate of gain in certain breeds of cattle (Kunkel, 1956). The investigation of the extent of the genetic control of biochemical patterns known to be affected by environmental forces is also of interest. The plasma or serum alkaline phosphatase in rats can be reduced by fasting 24 hours (Jackson, 1952) and this reduction is ameliorated by dietary protein and fat, and probably by supplemental choline (Tuba and Madsen, 1952). The serum alkaline phosphatase is increased in vitamin D deficient chicks (Motzok, 1950), decreased in manganese deficient chicks (Wiese et al., 1939), and increased with the administration of growth hormone to rats (Li el al., 1947). Breed differences as well as the considerable individual variation within each of three breeds of chickens were noted by Auchinachie and Emslie (1934). Common (1934, 1936) reported that the average phosphatase activity of sexually immature pullets is comparable with the average level in young cockerels. There was no significant difference between the phosphatase values of laying and those of
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 6, 2015
Plasma Alkaline Phosphatase Activity in Mature Inbred Chickens