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A Comparison of Methods of Testing Turkeys for Salmonella pullorum Infection*
A Comparison of Methods of Testing Turkeys for Salmonella pullorum Infection* F. W. GARLAND, JR., 1 A. R. WINTER AND E. R. AMIET 2
(Received for publication July 1, 1948)
S
INCE the stained antigen, rapid whole blood test, developed by Sha.ffer et al. (1931), has proven to be practical for testing chickens for Salmonella pullorum, there has been much interest in its efficiency for testing turkeys. Hinshaw et al. (1940), reported that commercial violet stained antigen, used for testing chickens by the whole blood test, was only about 50 percent as efficient as the tube agglutination test, used by the Research Workers in Animal Diseases (1933), for detecting S. pullorum infection in turkeys. Bushnell (1945) reported that the tube test, using 1:25 dilution was the most efficient in detecting S. pullorum infection, followed by the tube test at 1:50 dilution, the rapid serum test of Runnels et al. (1927), and the whole blood test using an experimental, turkey, violet, K-antigen, developed by the United States Bureau of Animal Industry. Gauger (1947) reported close agreement between the rapid wholeblood K-antigen and tube tests when the agglutination was complete at 1:25 or higher dilution. Corpron et al. (1947) found that agglutinins due to S. pullorum were detected earlier and for a longer time * This study was made possible by a grant from the Ohio State University Poultry Development Fund. 1 Formerly a Research Fellow. Now with The Ohio Poultry Improvement Association. 2 Formerly Turkey Supervisor, The Ohio Poultry Improvement Association.
by the tube test than by the whole-blood test using either K-antigen or turkey Redigen, a product of the Columbus Vaccine Company, Columbus, Ohio. Since the information on the comparative efficiency of the tube and wholeblood methods of testing turkeys for S. pullorum is limited and inconclusive, the object of this investigation has been to secure additional data. Whole blood tests, involving the use of K-antigen and turkey Redigen, have been compared with the tube test, at 1:25 and 1:50 dilutions, for detecting S. pullorum in turkeys. Birds of doubtful or variable reaction to the various tests have been slaughtered and cultured for S. pullorum. EXPERIMENTAL
The procedures used for testing were those listed in the National Turkey Improvement Plan (1947) unless otherwise stated in the trials. Trial 1. Five turkey breeding flocks, involving 1,665 birds were tested by the tube test at 1:25 and 1:50 dilutions. Two of the flocks, including 835 turkeys were also tested by the whole-blood method using regular violet chicken antigen, at the time blood samples were taken for the tube test. Three of the flocks, including 982 birds were tested by the wholeblood method with turkey K-antigen, supplied by the United States Bureau of Animal Industry. The serum remaining 63
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
Department of Poultry Science, Ohio State University, Columbus, Ohio
64
F. W. GARLAND, J R . , A. R. WINTER AND E. R. AMIET
ing, a sample was regarded as positive if it reacted to one or both tube tests (1:25 or 1:50 dilution) and one or both of the stained antigen tests on whole-blood or serum. A sample was regarded as negative, if it did not react to more than one of the four tests. The data are summarized in Table 2. We found only 182 reactors to the tube test at 1:25 dilution among the original 772 birds reported as reactors or suspi-
TABLE 1.—A comparison of methods of testing for "S. pullorum" infection
Flock
Number
Tube test 1:25
1 2 3 4 5
300 106 383 535 341
0 7+* 0 2+* 4+**
Chicken Violet
1:50
Blood
Serum
0 0
0
0
0
0
0 0 4+
Chicken Redigen
K-Antigen Blood
Serum
0
0
0 0
0 4+
Serum
4+
* Number of weak or doubtful reactors. ** Number of definite reactors.
Gauger (1947) and Corpron et al. (1947) have obtained similar results. Trial 2. From a flock of about 7,000, turkey breeders, 772 birds had been removed that had been called reactors or suspicious to the tube test at the 1:25 dilution by a commercial testing laboratory. The flock owner asked us to test the birds that had been removed, before they were sold, because he did not believe that infection was as bad as indicated by the commercial testing laboratory. These birds afforded a good opportunity to compare methods of testing. At the time blood samples were taken for the tube test, the whole-blood test was made with the use of turkey Redigen. The serum remaining from the tube test was used in place of whole-blood for making tests with K-antigen and turkey Redigen. The Kantigen tests were read within 3r minutes and those with turkey Redigen within 25 seconds. In the comparison of methods of test-
cious by the commercial laboratory. On checking with the laboratory for an explanation for the wide difference in results, it was learned that hemolyzed samples and those from which insufficient serum was obtainable had been improperly reported as reactors. The tube test at 1:25 dilution detected 87 percent of the positive birds while the whole-blood test with turkey Redigen detected 74 percent. The TABLE 2.—A comparison of methods of testing turkeys for "S. pullorum." Based on 210 positive and and 562 negative samples Number of known samples detected
Percent • efficiency
Tube 1:25 dilution
Positive 182 Negative 498
87 89
Turkey Redigen with whole blood
Positive 156 Negative 512
74 91
Turkey Redigen with serum
Positive 160 Negative 459
76 82
K-Antigen with serum
Positive 121 Negative 525
58 93
Method
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after the tube test was used in place of whole blood in making the stained antigen test on a number of the samples. In addition to chicken and turkey violet antigens, chicken Redigen was used on one flock. The data are summarized in Table 1. The percent of birds infected in all of the flocks was so low that little data were obtained for comparing methods. Weak reactors by the tube test at 1:25 dilution were not detected by the stained antigen tests.
65
PULLORUM TESTING METHODS TOE TURKEYS
the tube test at 1:25 dilution and 24 of them to the whole-blood test with turkey Redigen, at the time of slaughter. Based on isolation of S. pullorum, the tube test at the 1:25 dilution was 62 percent accurate and the whole-blood test with turkey Redigen was 65 percent. Hinshaw et al. (1940), Bushnell (1945), Gauger (1947), and Corpron et al. (1947) have also been unable to isolate S. pullorum from all birds that reacted to the tube test, especially at no greater titer than 1:25 dilution. The failure to isolate S. pullorum may be due to several factors: (1) that the birds are immune and no longer carriers, (2) to the failure to obtain infected tissues, (3) to non-specific reactions, (4) failure to recognize the organism. Coliforms were isolated from 20 of the 27 birds cultured where S. pullorum was not found. This group of organisms may have been responsible for the agglutination reaction since Bunyea and MacDonald (1942) and Edwards et al. (1943) have shown that cross-agglutination may occur between certain Coliform and Salmonella organisms. Trial 3. A group of 663 White Holland turkeys, that had been removed from a large breeding flock because they had shown varying degrees of reaction to the tube test at the 1:25 dilution, were retested by the tube test at 1:25 and 1:50 dilutions. At the time blood samples were taken for the tube test, the birds were tested by the whole-blood method, using both K-antigen and turkey Redigen. The data may be summarized as follows: Test Tube 1:25 dilution Turkey Redigen K-Antigen Tube 1:50 dilution Turkey Redigen K-Antigen
Number of Percent reactors accuracy* 309 100 320 103 210 68 216 320 210
100 148 97
* Assuming accuracy of tube test as 100 percent.
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turkey Redigen with serum detected 76 percent of the reactors while K-antigen with serum detected 58 percent. Dickinson et al. (1944) and Bushnell (1945) have obtained somewhat better correlation between the rapid test using serum in place of blood, and the tube test, than shown by the data from this trial. Sixty-four of the 210 birds called reactors by more than one of the tests did not give the same results when tested by all of the different methods. Forty of these birds were positive to the tube test at 1:25 dilution and 36 of them were also positive to the whole-blood test with turkey Redigen. If one assumes that the tube test at 1:25 dilution is 100 percent accurate, then the whole-blood test with turkey Redigen would be 90 percent accurate. The 64 birds that gave variable reactions to the different tests were slaughtered for market and cultured for the presence of Salmonellas at the time of evisceration. The viscera were removed aseptically and a portion of liver, heart, ovary and spleen from each bird were placed in a sterile Petri dish. The material was mascerated in a sterile mortar to which a little nutrient broth had been added. A few drops of the broth mixture were transferred to a tube of tetrathionate broth and incubated at 37°C. for 24 hours. Stroke plates were then made on (Bacto) SS, MacConkey's and bismuth sulfite agars. Colonies resembling those of the Salmonellas were tested on Kligler's iron agar and on sucrose mannitol agar slopes. Cultures giving positive Salmonella tests were used for making tube test antigens. They were then tested with known positive and negative S. pullorum serum. Those that were agglutinated by the positive serum but not by the negative serum, were regarded as S. pullorum. Salmonella pullorum was isolated from 37 of the 64 turkeys cultured. Twentythree of the positive birds had reacted to
66
F. W. GARLAND, J R . , A. R. WINTER AND E. R. AMIET
Test
Reactors
Suspicious
Negative
1st 2nd 3rd 4th
19 21 30 34
13 14 12 14
30 27 20 14
Apparently the infection spread among the birds during the three month period of observation. At the time of the final test, the birds were slaughtered for market. Tissues were obtained for culture at the time of evisceration. The same culture technique was used as described in Trial 2. The data are summarized in Table 3. S. pullorum was isolated from 22 of the birds, Escherichia TABLE 3.—Bacteria isolations from 62 White Holland turkeys Tube test reaction of birds Bacteria isolated
Salmonella pullorum only Salmonella pullorum and other bacteria Escherichia coli only Escherichi coli and other bacteria Streptococcus pyogenes Diplococcus Negative growth Total
Posi- Suspi- Nega- Total tive cious tive 10
4
1
15
5 6
2 4
0 9
7 18
3 7 3 0
0 3 1 1
0 1 2 1
3 11 6 2
34
14
14
62
coli from 21 and coccus bacteria from 17. No cultures were obtained from the other two. The various tests detected the following numbers of the 22 turkeys from which 5. pullorum was isolated and with the following accuracy:
Tube 1:25 dilution Tube 1:50 dilution Turkey Redigen K-antigen
Number Efficiency of detected test {percent) 21 95 16 73 22 100 15 68
This trial indicates that the tube agglutination test at 1:25 dilution and the whole-blood test with turkey Redigen check quite closely with the cultural findings. Since E. coli only was isolated from six birds that gave a positive reaction to the tube test at 1:25 dilution, tube test antigen was prepared from each of the cultures. They were tested with serum from known positive and negative pullorum reactor birds. Some agglutination of these antigens was produced by the positive serum but not by the negative serum. The presence of coliforms might therefore explain a positive S. pullorum test in some cases where the organism could not be found. The presence of coccus forms of bacteria in 10 of the samples might also account for S. pullorum agglutination where no Salmonellas were isolated. Hinshaw el al. (1942) have observed (nonspecific pullorum agglutination reactions) in turkeys from which coccus but not Salmonella bacteria were isolated. Trial 4. A group of 59 Bronze turkey hens were obtained from a flock of about 2,000 breeders. These birds had given variable or suspicious agglutination reactions to the tube test at 1:25 dilution and to the whole-blood test with turkey Redigen. The birds were slaughtered for market and blood obtained' at the time
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
The tube test at 1:25 dilution and the whole-blood test with turkey Redigen checked closely. The tube test at 1:50 dilution and the whole-blood test with Kantigen checked closely. Sixty-two of the 663 turkeys did not react the same to all of the tests and were purchased for further testing and culture. They were kept in confinement on litter and tested four times at 21 day intervals, using the tube test at 1:25 and 1:50 dilutions and the whole-blood test with turkey Redigen and K-antigen. The four tube tests at 1:25 dilution gave the following results:
PULLORUM TESTING METHODS FOR TURKEYS
TABLE 4.—Reactions of 59 turkeys classed as positive or doubtful reactors Number of reactors TVM
lest
Laboratory A Tube test—24 hour 1:25 dilution 1:50 dilution Tube test—48 hour 1:25 dilution 1:50 dilution Laboratory B Tube test—24 hour 1:25 dilution 1:50 dilution Tube test—48 hour 1:25 dilution 1:50 dilution Whole Blood Turkey Redigen K-Antigen Serum Turkey Redigen K-Antigen
Positive Suspicious incubation 37 28
4 5
41 32
2 6
18 12
11 9
40 32
5 7
31 30
11 9
27 22
9 7
incubation
incubation incubation
found after 48 hours incubation than after 24 hours, especially in our laboratory (Laboratory B). The two stained antigens gave about the same results when whole-blood was used. When serum was used turkey Redigen detected more reactors than K-antigen. Also, a few more reactors were detected with the stained antigens when whole-blood was used than when serum was used. S. pullorum was obtained from only six of the 59 birds cultured. Five of the six birds had reacted strongly to all of the agglutination tests. The sixth bird had been designated as a reactor by the tube tests, suspicious by the turkey Redigen tests and negative by the K-antigen tests. As judged by cultural findings in the trial, it would appear that our laboratory tube test at 1:50 dilution was more accurate than the other tests for detecting the presence of S. pullorum. It also appears that as far as detecting reactors is concerned (disregarding birds called reactors which are not), the tube test and the whole blood test with stained antigens; (K-antigen and turkey Redigen) compare favorably. SUMMARY
The data are summarized in Table 4. While the commercial laboratory (A) had called all 59 birds reactors or suspicious on the previous test, only 41 were so classified by the same laboratory at the time of autopsy. We found 18 reactors, only 7 of which were classified as complete at 1:25 dilution, and 11 others that might be classified as suspicious. As might be expected, both laboratories found more reactors at 1:25 than at 1:50 dilution. The tube tests were read after 24 hours incubation at 37°C. and again after 48 hours incubation. More reactors were
Several hundred turkey breeders have been tested by the tube test at 1:25 and 1:50 dilutions and by the whole-blood test with turkey Redigen and K-Antigen. One hundred and eighty-five birds that gave doubtful or variable reactions to the different tests were slaughtered and cultured for the presence of Salmonella pullorum. The tube test, with complete agglutination at 1:25 dilution, and the whole-blood test, with turkey Redigen, compared favorably for detecting carriers of S. pullorum. K-antigen was slightly less sensitive
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for the whole-blood test with K-antigen (Lot C-5) and turkey Redigen (Lot 424) and the tube test at 1:25 and 1:50 dilutions. Serum remaining from the tube test was used for testing with the two stained antigens. At the time of evisceration tissues were obtained for culturing for the presence of 5. pullorum. The same procedure was used as in Trial 2.
67
68
F. W. GARLAND, J R . , A. R. WINTER AND E. R. AMIET
than turkey Redigen for detecting S. pullorum carriers. Escherichia coli infection was demonstrated as a cause of a positive pullorum reaction in some cases in which S. pullorum was not isolated.
Bunyea, H., and A. D. MacDonald, 1942. The pathogenicity of Aerobacter aerogenes and Escherichia acidi lactici for turkeys and their response to the agglutination test for pullorum disease. Poultry Sci. 21: 306-310. Bushnell, L. D., 1945. Pullorum testing of turkeys. Poultry Sci. 24: 208-211. Corpron, Ruth, J- A. Bivens, and H. J. Stafseth, 1947. Pullorum disease studies in turkeys. I. Antibody production following intravenous and oral infection, relative value of tube and wholeblood plate agglutination tests, pen contagion and duration of carrier state. Poultry Sci. 26:340-351. Dickinson, E. M., A. S. Rosenwald, and D. R. Morrill, 1944. Comparison of the tube and rapid serum agglutination tests for the detection of pullorum disease in turkeys. Ore. Agr. Exp. Sta. Tech. Bui. 6. Edwards, P. R., W. B. Cherry, and D. W. Bruner, 1943. Further studies on coliform bacteria sero-
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
REFERENCES
logically related to the genus Salmonella. Jour. Inf. Dis. 73: 229-238. Gauger, H. C , 1947. Comparison of the rapid wholeblood K-antigen and the tube agglutination test for the detection of pullorum disease in turkeys. Poultry Sci. 26: 229-233. Hinshaw, W. R., E. E. Jones, J. F. Harr, and W. E. Niemeyer, 1940. Comparison of the tube and the whole blood tests for pullorum disease of turkeys. Cornell Vet. 30: 30-38. Hinshaw, W. R., E. McNeil, and T. J. Taylor, 1942. Four years progress in eradication of pullorum disease from turkey flocks. Proc. 46th Ann. Meeting U. S. Livestock Sanitary Assoc, pp. 224-237. The National Turkey Improvement Plan, 1947. TJ. S. Dept. Agr. Misc. Pub. 555. Research Workers in Animal Diseases, 1933. Report of the conference of official research workers in animal diseases. II. Serological diagnosis of pullorum disease. Jour. Amer. Vet. Med. Assoc. 82: 488-490. Runnells, R. A., C. J. Coon, H. Farley, and F. Thorp, 1927. An application of the rapidmethod agglutination test to the diagnosis of bacillary white diarrhea infection. Jour. Amer. Vet. Med. Assoc. 70: 660-662. Shaffer, J. M., A. D. MacDonald, W. J. Hall, and H. Bunyea, 1931. A stained antigen for the rapid whole blood test for pullorum disease. Jour. Amer. Vet. Med. Assoc. 79: 236-240.
(Received for publication July 1, 1948)
S
INCE the stained antigen, rapid whole blood test, developed by Sha.ffer et al. (1931), has proven to be practical for testing chickens for Salmonella pullorum, there has been much interest in its efficiency for testing turkeys. Hinshaw et al. (1940), reported that commercial violet stained antigen, used for testing chickens by the whole blood test, was only about 50 percent as efficient as the tube agglutination test, used by the Research Workers in Animal Diseases (1933), for detecting S. pullorum infection in turkeys. Bushnell (1945) reported that the tube test, using 1:25 dilution was the most efficient in detecting S. pullorum infection, followed by the tube test at 1:50 dilution, the rapid serum test of Runnels et al. (1927), and the whole blood test using an experimental, turkey, violet, K-antigen, developed by the United States Bureau of Animal Industry. Gauger (1947) reported close agreement between the rapid wholeblood K-antigen and tube tests when the agglutination was complete at 1:25 or higher dilution. Corpron et al. (1947) found that agglutinins due to S. pullorum were detected earlier and for a longer time * This study was made possible by a grant from the Ohio State University Poultry Development Fund. 1 Formerly a Research Fellow. Now with The Ohio Poultry Improvement Association. 2 Formerly Turkey Supervisor, The Ohio Poultry Improvement Association.
by the tube test than by the whole-blood test using either K-antigen or turkey Redigen, a product of the Columbus Vaccine Company, Columbus, Ohio. Since the information on the comparative efficiency of the tube and wholeblood methods of testing turkeys for S. pullorum is limited and inconclusive, the object of this investigation has been to secure additional data. Whole blood tests, involving the use of K-antigen and turkey Redigen, have been compared with the tube test, at 1:25 and 1:50 dilutions, for detecting S. pullorum in turkeys. Birds of doubtful or variable reaction to the various tests have been slaughtered and cultured for S. pullorum. EXPERIMENTAL
The procedures used for testing were those listed in the National Turkey Improvement Plan (1947) unless otherwise stated in the trials. Trial 1. Five turkey breeding flocks, involving 1,665 birds were tested by the tube test at 1:25 and 1:50 dilutions. Two of the flocks, including 835 turkeys were also tested by the whole-blood method using regular violet chicken antigen, at the time blood samples were taken for the tube test. Three of the flocks, including 982 birds were tested by the wholeblood method with turkey K-antigen, supplied by the United States Bureau of Animal Industry. The serum remaining 63
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
Department of Poultry Science, Ohio State University, Columbus, Ohio
64
F. W. GARLAND, J R . , A. R. WINTER AND E. R. AMIET
ing, a sample was regarded as positive if it reacted to one or both tube tests (1:25 or 1:50 dilution) and one or both of the stained antigen tests on whole-blood or serum. A sample was regarded as negative, if it did not react to more than one of the four tests. The data are summarized in Table 2. We found only 182 reactors to the tube test at 1:25 dilution among the original 772 birds reported as reactors or suspi-
TABLE 1.—A comparison of methods of testing for "S. pullorum" infection
Flock
Number
Tube test 1:25
1 2 3 4 5
300 106 383 535 341
0 7+* 0 2+* 4+**
Chicken Violet
1:50
Blood
Serum
0 0
0
0
0
0
0 0 4+
Chicken Redigen
K-Antigen Blood
Serum
0
0
0 0
0 4+
Serum
4+
* Number of weak or doubtful reactors. ** Number of definite reactors.
Gauger (1947) and Corpron et al. (1947) have obtained similar results. Trial 2. From a flock of about 7,000, turkey breeders, 772 birds had been removed that had been called reactors or suspicious to the tube test at the 1:25 dilution by a commercial testing laboratory. The flock owner asked us to test the birds that had been removed, before they were sold, because he did not believe that infection was as bad as indicated by the commercial testing laboratory. These birds afforded a good opportunity to compare methods of testing. At the time blood samples were taken for the tube test, the whole-blood test was made with the use of turkey Redigen. The serum remaining from the tube test was used in place of whole-blood for making tests with K-antigen and turkey Redigen. The Kantigen tests were read within 3r minutes and those with turkey Redigen within 25 seconds. In the comparison of methods of test-
cious by the commercial laboratory. On checking with the laboratory for an explanation for the wide difference in results, it was learned that hemolyzed samples and those from which insufficient serum was obtainable had been improperly reported as reactors. The tube test at 1:25 dilution detected 87 percent of the positive birds while the whole-blood test with turkey Redigen detected 74 percent. The TABLE 2.—A comparison of methods of testing turkeys for "S. pullorum." Based on 210 positive and and 562 negative samples Number of known samples detected
Percent • efficiency
Tube 1:25 dilution
Positive 182 Negative 498
87 89
Turkey Redigen with whole blood
Positive 156 Negative 512
74 91
Turkey Redigen with serum
Positive 160 Negative 459
76 82
K-Antigen with serum
Positive 121 Negative 525
58 93
Method
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
after the tube test was used in place of whole blood in making the stained antigen test on a number of the samples. In addition to chicken and turkey violet antigens, chicken Redigen was used on one flock. The data are summarized in Table 1. The percent of birds infected in all of the flocks was so low that little data were obtained for comparing methods. Weak reactors by the tube test at 1:25 dilution were not detected by the stained antigen tests.
65
PULLORUM TESTING METHODS TOE TURKEYS
the tube test at 1:25 dilution and 24 of them to the whole-blood test with turkey Redigen, at the time of slaughter. Based on isolation of S. pullorum, the tube test at the 1:25 dilution was 62 percent accurate and the whole-blood test with turkey Redigen was 65 percent. Hinshaw et al. (1940), Bushnell (1945), Gauger (1947), and Corpron et al. (1947) have also been unable to isolate S. pullorum from all birds that reacted to the tube test, especially at no greater titer than 1:25 dilution. The failure to isolate S. pullorum may be due to several factors: (1) that the birds are immune and no longer carriers, (2) to the failure to obtain infected tissues, (3) to non-specific reactions, (4) failure to recognize the organism. Coliforms were isolated from 20 of the 27 birds cultured where S. pullorum was not found. This group of organisms may have been responsible for the agglutination reaction since Bunyea and MacDonald (1942) and Edwards et al. (1943) have shown that cross-agglutination may occur between certain Coliform and Salmonella organisms. Trial 3. A group of 663 White Holland turkeys, that had been removed from a large breeding flock because they had shown varying degrees of reaction to the tube test at the 1:25 dilution, were retested by the tube test at 1:25 and 1:50 dilutions. At the time blood samples were taken for the tube test, the birds were tested by the whole-blood method, using both K-antigen and turkey Redigen. The data may be summarized as follows: Test Tube 1:25 dilution Turkey Redigen K-Antigen Tube 1:50 dilution Turkey Redigen K-Antigen
Number of Percent reactors accuracy* 309 100 320 103 210 68 216 320 210
100 148 97
* Assuming accuracy of tube test as 100 percent.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
turkey Redigen with serum detected 76 percent of the reactors while K-antigen with serum detected 58 percent. Dickinson et al. (1944) and Bushnell (1945) have obtained somewhat better correlation between the rapid test using serum in place of blood, and the tube test, than shown by the data from this trial. Sixty-four of the 210 birds called reactors by more than one of the tests did not give the same results when tested by all of the different methods. Forty of these birds were positive to the tube test at 1:25 dilution and 36 of them were also positive to the whole-blood test with turkey Redigen. If one assumes that the tube test at 1:25 dilution is 100 percent accurate, then the whole-blood test with turkey Redigen would be 90 percent accurate. The 64 birds that gave variable reactions to the different tests were slaughtered for market and cultured for the presence of Salmonellas at the time of evisceration. The viscera were removed aseptically and a portion of liver, heart, ovary and spleen from each bird were placed in a sterile Petri dish. The material was mascerated in a sterile mortar to which a little nutrient broth had been added. A few drops of the broth mixture were transferred to a tube of tetrathionate broth and incubated at 37°C. for 24 hours. Stroke plates were then made on (Bacto) SS, MacConkey's and bismuth sulfite agars. Colonies resembling those of the Salmonellas were tested on Kligler's iron agar and on sucrose mannitol agar slopes. Cultures giving positive Salmonella tests were used for making tube test antigens. They were then tested with known positive and negative S. pullorum serum. Those that were agglutinated by the positive serum but not by the negative serum, were regarded as S. pullorum. Salmonella pullorum was isolated from 37 of the 64 turkeys cultured. Twentythree of the positive birds had reacted to
66
F. W. GARLAND, J R . , A. R. WINTER AND E. R. AMIET
Test
Reactors
Suspicious
Negative
1st 2nd 3rd 4th
19 21 30 34
13 14 12 14
30 27 20 14
Apparently the infection spread among the birds during the three month period of observation. At the time of the final test, the birds were slaughtered for market. Tissues were obtained for culture at the time of evisceration. The same culture technique was used as described in Trial 2. The data are summarized in Table 3. S. pullorum was isolated from 22 of the birds, Escherichia TABLE 3.—Bacteria isolations from 62 White Holland turkeys Tube test reaction of birds Bacteria isolated
Salmonella pullorum only Salmonella pullorum and other bacteria Escherichia coli only Escherichi coli and other bacteria Streptococcus pyogenes Diplococcus Negative growth Total
Posi- Suspi- Nega- Total tive cious tive 10
4
1
15
5 6
2 4
0 9
7 18
3 7 3 0
0 3 1 1
0 1 2 1
3 11 6 2
34
14
14
62
coli from 21 and coccus bacteria from 17. No cultures were obtained from the other two. The various tests detected the following numbers of the 22 turkeys from which 5. pullorum was isolated and with the following accuracy:
Tube 1:25 dilution Tube 1:50 dilution Turkey Redigen K-antigen
Number Efficiency of detected test {percent) 21 95 16 73 22 100 15 68
This trial indicates that the tube agglutination test at 1:25 dilution and the whole-blood test with turkey Redigen check quite closely with the cultural findings. Since E. coli only was isolated from six birds that gave a positive reaction to the tube test at 1:25 dilution, tube test antigen was prepared from each of the cultures. They were tested with serum from known positive and negative pullorum reactor birds. Some agglutination of these antigens was produced by the positive serum but not by the negative serum. The presence of coliforms might therefore explain a positive S. pullorum test in some cases where the organism could not be found. The presence of coccus forms of bacteria in 10 of the samples might also account for S. pullorum agglutination where no Salmonellas were isolated. Hinshaw el al. (1942) have observed (nonspecific pullorum agglutination reactions) in turkeys from which coccus but not Salmonella bacteria were isolated. Trial 4. A group of 59 Bronze turkey hens were obtained from a flock of about 2,000 breeders. These birds had given variable or suspicious agglutination reactions to the tube test at 1:25 dilution and to the whole-blood test with turkey Redigen. The birds were slaughtered for market and blood obtained' at the time
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on May 22, 2015
The tube test at 1:25 dilution and the whole-blood test with turkey Redigen checked closely. The tube test at 1:50 dilution and the whole-blood test with Kantigen checked closely. Sixty-two of the 663 turkeys did not react the same to all of the tests and were purchased for further testing and culture. They were kept in confinement on litter and tested four times at 21 day intervals, using the tube test at 1:25 and 1:50 dilutions and the whole-blood test with turkey Redigen and K-antigen. The four tube tests at 1:25 dilution gave the following results:
PULLORUM TESTING METHODS FOR TURKEYS
TABLE 4.—Reactions of 59 turkeys classed as positive or doubtful reactors Number of reactors TVM
lest
Laboratory A Tube test—24 hour 1:25 dilution 1:50 dilution Tube test—48 hour 1:25 dilution 1:50 dilution Laboratory B Tube test—24 hour 1:25 dilution 1:50 dilution Tube test—48 hour 1:25 dilution 1:50 dilution Whole Blood Turkey Redigen K-Antigen Serum Turkey Redigen K-Antigen
Positive Suspicious incubation 37 28
4 5
41 32
2 6
18 12
11 9
40 32
5 7
31 30
11 9
27 22
9 7
incubation
incubation incubation
found after 48 hours incubation than after 24 hours, especially in our laboratory (Laboratory B). The two stained antigens gave about the same results when whole-blood was used. When serum was used turkey Redigen detected more reactors than K-antigen. Also, a few more reactors were detected with the stained antigens when whole-blood was used than when serum was used. S. pullorum was obtained from only six of the 59 birds cultured. Five of the six birds had reacted strongly to all of the agglutination tests. The sixth bird had been designated as a reactor by the tube tests, suspicious by the turkey Redigen tests and negative by the K-antigen tests. As judged by cultural findings in the trial, it would appear that our laboratory tube test at 1:50 dilution was more accurate than the other tests for detecting the presence of S. pullorum. It also appears that as far as detecting reactors is concerned (disregarding birds called reactors which are not), the tube test and the whole blood test with stained antigens; (K-antigen and turkey Redigen) compare favorably. SUMMARY
The data are summarized in Table 4. While the commercial laboratory (A) had called all 59 birds reactors or suspicious on the previous test, only 41 were so classified by the same laboratory at the time of autopsy. We found 18 reactors, only 7 of which were classified as complete at 1:25 dilution, and 11 others that might be classified as suspicious. As might be expected, both laboratories found more reactors at 1:25 than at 1:50 dilution. The tube tests were read after 24 hours incubation at 37°C. and again after 48 hours incubation. More reactors were
Several hundred turkey breeders have been tested by the tube test at 1:25 and 1:50 dilutions and by the whole-blood test with turkey Redigen and K-Antigen. One hundred and eighty-five birds that gave doubtful or variable reactions to the different tests were slaughtered and cultured for the presence of Salmonella pullorum. The tube test, with complete agglutination at 1:25 dilution, and the whole-blood test, with turkey Redigen, compared favorably for detecting carriers of S. pullorum. K-antigen was slightly less sensitive
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for the whole-blood test with K-antigen (Lot C-5) and turkey Redigen (Lot 424) and the tube test at 1:25 and 1:50 dilutions. Serum remaining from the tube test was used for testing with the two stained antigens. At the time of evisceration tissues were obtained for culturing for the presence of 5. pullorum. The same procedure was used as in Trial 2.
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F. W. GARLAND, J R . , A. R. WINTER AND E. R. AMIET
than turkey Redigen for detecting S. pullorum carriers. Escherichia coli infection was demonstrated as a cause of a positive pullorum reaction in some cases in which S. pullorum was not isolated.
Bunyea, H., and A. D. MacDonald, 1942. The pathogenicity of Aerobacter aerogenes and Escherichia acidi lactici for turkeys and their response to the agglutination test for pullorum disease. Poultry Sci. 21: 306-310. Bushnell, L. D., 1945. Pullorum testing of turkeys. Poultry Sci. 24: 208-211. Corpron, Ruth, J- A. Bivens, and H. J. Stafseth, 1947. Pullorum disease studies in turkeys. I. Antibody production following intravenous and oral infection, relative value of tube and wholeblood plate agglutination tests, pen contagion and duration of carrier state. Poultry Sci. 26:340-351. Dickinson, E. M., A. S. Rosenwald, and D. R. Morrill, 1944. Comparison of the tube and rapid serum agglutination tests for the detection of pullorum disease in turkeys. Ore. Agr. Exp. Sta. Tech. Bui. 6. Edwards, P. R., W. B. Cherry, and D. W. Bruner, 1943. Further studies on coliform bacteria sero-
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REFERENCES
logically related to the genus Salmonella. Jour. Inf. Dis. 73: 229-238. Gauger, H. C , 1947. Comparison of the rapid wholeblood K-antigen and the tube agglutination test for the detection of pullorum disease in turkeys. Poultry Sci. 26: 229-233. Hinshaw, W. R., E. E. Jones, J. F. Harr, and W. E. Niemeyer, 1940. Comparison of the tube and the whole blood tests for pullorum disease of turkeys. Cornell Vet. 30: 30-38. Hinshaw, W. R., E. McNeil, and T. J. Taylor, 1942. Four years progress in eradication of pullorum disease from turkey flocks. Proc. 46th Ann. Meeting U. S. Livestock Sanitary Assoc, pp. 224-237. The National Turkey Improvement Plan, 1947. TJ. S. Dept. Agr. Misc. Pub. 555. Research Workers in Animal Diseases, 1933. Report of the conference of official research workers in animal diseases. II. Serological diagnosis of pullorum disease. Jour. Amer. Vet. Med. Assoc. 82: 488-490. Runnells, R. A., C. J. Coon, H. Farley, and F. Thorp, 1927. An application of the rapidmethod agglutination test to the diagnosis of bacillary white diarrhea infection. Jour. Amer. Vet. Med. Assoc. 70: 660-662. Shaffer, J. M., A. D. MacDonald, W. J. Hall, and H. Bunyea, 1931. A stained antigen for the rapid whole blood test for pullorum disease. Jour. Amer. Vet. Med. Assoc. 79: 236-240.