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Use of the Spot Test in the Diagnosis of Pullorum Disease*,1
Use of the Spot Test in the Diagnosis of Pullorum Disease*1 J. E. WILLIAMS Veterinary Diagnosis Laboratory, University of Minnesota, St. Paul (Received for publication July 17, 1950)
T
* Paper No. 2573 scientific journal series, Minnesota Agricultural Experiment Station; taken from a thesis to be submitted to the graduate school of the University of Minnesota in partial fulfillment of the requirements for the degree of doctor of philosophy. 1 The author gratefully acknowledges the assistance of Miss Florence Jones. The investigations reported in this paper were supported in part by a grant-in-aid from the International Baby Chick Association.
has at his disposal antigens prepared from the standard or variant strain alone as well as polyvalent antigen composed of a mixture of standard and variant strains. Since, in most cases, it has not been practical to employ a double testing program, the latter antigen has often found favor in those flocks where both standard and variant types of pullorum disease have been encountered. A second factor in the effective application of the pullorum test is the problem of suspicious or so-called "pin-point" reactors. In this category are included those birds whose blood gives a late, very fine agglutination on the rapid whole blood plate test and a fine, easily dispersed, often low-titered reaction on the macroscopic tube test. These reactions may be encountered in certain flocks from year to year or they may be demonstrable for only a short period of time. In any event, they present a definite problem, especially when they are encountered in large numbers. While it is recognized that several factors may be involved in these atypical reactions, studies conducted so far suggest that cross-reacting agglutinins for S. pullorum occur in low or occasionally high titer in the blood of such birds. Investigations by Garrard et al. (1946, 1947), Carpenter et al. (1947) and Burton and Garrard (1948), have indicated that subclinical infections with coliforms, micrococci (staphylococci), enterococci, and other organisms may initiate these suspicious reactions.
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HERE are certain factors that tend to interfere with the effective application of the agglutination test employed for the diagnosis of pullorum disease in chickens and turkeys. As the testing program has decreased the incidence of infected flocks, two of these factors have become of special concern to those engaged in the control of pullorum disease. First, there is the recent recognition by Younie (1941), Edwards and Bruner (1946) and others that Salmonella pullorum is subject to antigenic variation, giving rise to so-called standard and variant types. While the variant type of pullorum disease has been detected in various parts of the United States and Canada, more extensive serological typing is necessary to determine the true extent of variant infection. Investigations in recent years have established that either type of the disease may escape detection if an antigen prepared from the opposite type is used in testing. This fact has prompted a reconsideration of the antigen problem and has resulted in the appearance of a variety of antigens on the market. The tester now
126
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WILLIAMS
The variant and polyvalent pullorum antigens seem to show a greater affinity for cross-reacting agglutinins t h a n the standard antigen. Therefore, the former antigens are usually employed only in those flocks where the variant type of pullorum disease has been encountered. In view of the problem, efforts have been made to eliminate suspicious reac-
employed in screening out questionable reactors. T h e test and the results of investigations of its application to the diagnosis of pullorum disease in chickens and turkeys are discussed in this paper. EQUIPMENT
The following equipment is required for conducting the test:
FIG. 1. 1. A positive spot test using polyvalent stained antigen. Note that the antigen has been fixed in the center of the drop. 2. A negative test using polyvalent antigen. The antigen is distributed throughout the drop. 3. A negative test with a suspicious serum sample using polyvalent antigen.
tors through the development of more specific antigens for testing. However, the problem has persisted, and large numbers of suspicious reactors have been submitted to culture with negative results. There is no reliable method available for screening out suspicious reactors with any degree of certainty. T h e use by Castaneda (1950) of a unique and simple spot test (surface fixation test) for the detection of brucellosis, prompted our investigation of the application of this test to the diagnosis of pullorum disease. I t was believed t h a t because of the nature of the test it might be
1 loop (4.5 mm. internal diameter, 20 gauge wire) 1 loop (2 mm. internal diameter, 24 gauge wire) Filter paper (Schleicher & Schuell, No. 589, White Ribbon, or an equivalent moderately retentive grade) 2 petri dish covers containing physiological saline Pullorum stained antigen (4% cells) Serum samples to be tested PROCEDURE 1. Place a sheet of filter paper on top of a wire test tube rack. Mark the filter paper with the sample number or indicate in some other way the order of the samples to be tested. 2. Carefully place a drop of the serum on the filter paper using the 4.5 mm. loop. The loop should not be bulging with the serum but
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
4t|
SPOT TEST IN PULLORUM DIAGNOSIS
In conducting the test only one serum sample should be tested at a time and should be deposited on the filter paper immediately before the test is to be conducted. The loop should be rinsed in saline between each sample tested. Hemolyzed or partially jelled samples are unsuitable for testing. It is essential that steps 2, 3, and 4 be conducted in rapid succession. The antigen used is a doubly concentrated preparation of the usual crystal violet stained antigen prepared according to the specifications of the U. S. Bureau of Animal Industry, and employed in conducting the rapid whole blood test for pullorum disease. An antigen stained with aqueous hematoxylin proved inferior to the crystal violet antigen. The mechanism of the spot test seems to be a simple fixation of the antigen in the center of the drop of serum, due to a rapid and strong union of antigen and antibody, in the case of a positive serum sample. With a negative serum sample no such union takes place and the stained organisms are free to spread out through the area when the saline is added. Partial or weakly reacting agglutinins encountered in suspicious reactors are, in most cases, not capable of combining with the
127
antigen particles, in so short a period of time, or combine so loosely that the antigen particles are readily washed away with the saline (Figure 1; 3). RESULTS
At the time when this test came to our attention a group of twenty-two adult female chickens were being studied for agglutinin titers with pullorum antigens. These birds were accumulated from field flocks in which suspicious reactors had been encountered when the birds were tested with polyvalent pullorum antigen, using the whole blood plate test. The birds had been under observation for a period of eight months and had been tested at frequent intervals during this time, using a variety of plate and tube antigens. Sixteen of the birds were classified as suspicious reactors. Only two of the chickens were believed to be infected with pullorum disease as revealed by hightitered, coarse agglutination reactions and positive egg culture. Four of the birds were entirely negative with all the antigens that were used. When the serums of these birds were tested with the spot test using polyvalent pullorum antigen only two samples revealed positive tests and these were from the two birds that had previously been classified as infected. The organs of each individual bird were submitted to careful bacteriological examination using several different mediums in an effort to recover any organisms which they might harbor. S. pullorum was not isolated from any of the chickens except the two that revealed the positive reactions as noted above. It is of interest to mention an Escherichia coli organism isolated from the liver of bird No. 3, a suspicious reactor with variant and polyvalent antigens. A crystal violet stained antigen was prepared from this organism and used in the spot test
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
should contain enough so that when it spreads it will cover an area about 12 mm. in diameter. 3. Using the 2 mm. loop immediately place 1 bulging loopful of the stained antigen on the center of the drop of serum. It is most essential that the antigen be slowly and carefully lowered onto the drop of serum while holding the loop almost parallel to the paper. 4. Immediately deposit two or three drops of saline on top of the antigen drop using the 2 mm. loop. The saline will spread at once through the antigen-serum mixture. 5. Allow the test to dry slightly and read. Positive: The antigen will remain in the center of the drop (Figure 1; 1). Negative: The antigen will spread out through the drop to color the whole area (Figure 1; 2, 3).
-
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#
:;:
TABLE 1.—Results of spot test with serum samples of thirty-two chickens
Agglutination
Spot test*
Culture results
No. of birds
Suspicious Suspicious Suspicious Suspicious Positive Negative Positive Positive Negative Positive Negative
Negative Negative Positive Positive Positive Negative Positive Negative Positive Negative Negative
Negative S. pullorum S. pullorum Negative S. pullorum Negative Negative S. pullorum S. pullorum Negative S. pullorum
14 0 0 1 10
s 2
0 0 0 0
* The readings noted were obtained with polyvalent stained antigen except for one infected bird that would have been classified as negative if standard antigen had not been used. Suspicious agglutination—Those samples revealing a slow, finely granular agglutination with the rapid whole blood plate test and/or a low or occasionally high-titered, easily dispersed reaction with incomplete supernatant clearing with the tube test. Positive agglutination—Rapid, coarse agglutination with the plate test and firm, coarse agglutination with clear supernatant at a dilution of 1-25 or higher with the tube test.
with the serum of bird No. 3. A positive reaction resulted from this combination; however, a negative test was obtained when a variant pullorum serum was tested using this antigen (Figure 2). These preliminary results indicated that the spot test might be useful in demonstrating the affinity of pullorum antigens for their homologous agglutinins, while eliminating any partial attraction for cross-reacting agglutinins. Serum samples from a group of thirtytwo chickens were subsequently tested. These chickens were selected at random from field flocks and included positive, negative, and suspicious reactors. After a period of observation, the birds were submitted to complete bacteriological examination for S. pullorum. Results obtained with the spot test on examining the serums of this group of chickens, are summarized in Table 1. It is
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FIG. 2. 1. Positive spot test of the serum of a suspicious reactor (No. 3) using a stained antigen prepared from an E. coli culture isolated from the liver of the same bird. 2. Negative test of the serum of a variant infected chicken using the E. coli stained antigen.
SPOT TEST IN PULLORUM DIAGNOSIS
notable that almost one-half of the birds were suspicious reactors with the agglutination test. All of these birds gave negative readings on the spot test and did not yield S. pullorum on culture. The spot test detected every bird that gave a typical positive reaction with the agglutination test. S. pullorum was isolated from
V
alone had been used (Figure 3). All of the S. pullorum cultures isolated from these chickens were of the standard type. In conducting the spot tests on this group of birds the polyvalent antigen detected all of the positive birds except one. However, the serum of this bird gave a very markedly positive spot test with
SV
m I FIG. 3. 1, 2, 3. Tests of the serum of a standard infected chicken using standard (S), variant (V), and polyvalent (SV) stained antigens.
six of these birds. In no case were positive cultural results obtained with negative spot test results. Two birds were positive on all tests but did not yield S. pullorum. In conducting the agglutination tests noted in Table 1 both tube and plate antigens of standard, variant, and polyvalent types were used. Tube tests were incubated at 37 degrees C. for 24 hours. The results with these individual antigens have not been included because of the space that would be required. However, it should be pointed out that the majority of the suspicious reactions occurred with the variant and polyvalent antigens. Two of the positive birds would have been classified as negative if variant antigen
standard antigen while with variant antigen it was negative. Each year suspicious reactors are encountered in testing turkeys for pullorum disease. Here again these reactions are more frequently encountered with variant and polyvalent antigens. Spot test results correlated with agglutination and cultural results on a group of 104 turkeys are summarized in Table 2. Most of these turkeys were accumulated from various flocks as suspicious reactors during the 1949 testing season. At the time of bacteriological examination their serums were preserved with merthiolate and refrigerated. It is significant that approximately fifty
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s
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percent of these birds while suspicious reactors with the agglutination test, were negative with the spot test and negative on culture. Three birds classified as suspicious agglutination reactors yielded S. pullorum on culture. One of these birds was positive with the spot test. Two of TABLE 2.—Results of spot test with serum samples of 104 turkeys Spot testf
Culture results
No. of birds
Suspicious Suspicious Suspicious Suspicious Suspicious Positive Negative Positive Positive Negative Positive Negative
Negative Negative Positive Positive Negative Positive Negative Positive Negative Positive Negative Negative
Negative S. pullorum S. pullorum Negative Paratyphoid S. pullorum Negative Negative S. pullorum S. pullorum Negative S. pullorum
50 2 1 5 2 9 28 4 0 0 2 1
* Tube tests were conducted using standard, variant, and polyvalent antigens. Tests were incubated at 37 degrees C. for 24 hours. t The results were obtained with polyvalent stained antigen. Suspicious agglutination—Indicated by a low or high-titered, fine, easily dispersed reaction with incomplete supernatant clearing on the tube test. Positive agglutination—Firm, coarse agglutination with clear supernatant at a dilution of 1-25 or higher with the tube test.
them would have been missed on the spot test. Five birds were suspicious agglutination reactors with positive spot tests and negative culture results. Two birds supicious with the 'agglutination test were negative on the spot test and yielded paratyphoid organisms on culture. Typing results on these paratyphoid cultures are not yet available. All of the S. pullorum cultures isolated from these turkeys were of the standard type. Fifty-five other turkey serum samples suspicious with the agglutination test were subsequently tested. The majority of these samples were negative with the spot test. Nine other birds revealing coarsely
DISCUSSION
The results presented above indicate that the spot test may serve as a useful adjunct to the agglutination test in the diagnosis of pullorum disease in chickens and turkeys. In testing typical pullorum reactors the sensitivity of the spot test correlated very closely with that of the agglutination test. The intensity of the fixation reaction in the spot test seems to be a function of the quantitative as well as the qualitative antibody content of the serum, and this may vary in degree depending upon the sample tested. Occasionally suspicious serum samples are encountered which reveal weakly positive spot test reactions. Such samples usually show an agglutination titer of 1-50 or even higher with pullorum antigens. Dilution of serum samples with saline tends to interfere to a certain extent with the mechanics of the test; however, it has been possible to show that undiluted samples typically positive with the tube agglutination test at a dilution of 1-25 give positive spot tests. In most cases, a polyvalent pullorum antigen used in the spot test to examine serums of known agglutinin content has proved equally effective in detecting either the standard or variant type of pullorum disease. However, in a few cases it was of advantage to use both the standard and variant antigens in order to demonstrate better the degree of reaction. In a number of instances an antigen prepared from the standard type of S. pullorum would not have detected a variant-inflected bird and vice-versa. The rapidity and ease with which the
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Agglutination*
positive tube agglutination tests were strongly positive spot test reactors. None of these birds have yet been submitted to bacteriological examination.
NEWS AND NOTES
SUMMARY The application of the spot test to the diagnosis of pullorum infection in chickens and turkeys is discussed. The method of conducting the test is presented. Results are cited which indicate that the test may be useful as an adjunct to the agglutination test, especially in flocks where suspicious reactors are a problem. REFERENCES Burton, W. H., and E. H. Garrard, 1948. Non-
pullorum agglutination reactions. IV. Reactions with pullorum antigen from fowl inoculated with coliform types. Can. J. Comp. Med. 12: 20-25. Carpenter, J. A., W. H. Burton and E. H. Garrard, 1947. Non-pullorum agglutination reactions. III. Reactions with pullorum antigen from fowl inoculated with an enterococcus. Can J. Comp. Med. 11:163-168. Castaneda, M. R., 1950. Surface fixation. A new method of detecting certain immunological reactions. Proc. Soc. Exp. Biol. Med. 73: 46-49. Edwards, P. R., and D. W. Bruner, 1946. Form variation in Salmonella pullorum and its relation to X strains. Cornell Vet. 36: 318-324. Garrard, E. H., L. A. McDermott, W. H . Burton and J. A. Carpenter, 1946. Non-specific pullorum agglutination reactions. I. Preliminary observations on fowl exhibiting non-specific reactions over an extended period. Can. J.'Comp. Med. 10: 342-347. Garrard, E. H., W. H. Burton, J. A. Carpenter and L. A. McDermott, 1947. Non-specific pullorum agglutination reactions. II. Post mortem studies on fowl exhibiting non-specific reactions over an extended period. Can. J. Comp. Med. 11: 102107. Younie, A. R., 1941. Fowl infection like pullorum disease. Can. J. Comp. Med. 5:164-167.
News and Notes {Continued from page 124)
egg products, and a close-up view of the Poultry and Egg National Board's retail training program. Food and Drug, and Public Health officials will participate in the Good Housekeeping Clinic on Monday afternoon when the Institute's new edition of Suggested Sanitary Standards for Poultry Plants will make its debut. Allan B. Kline, President of the American Farm Bureau Federation, will speak at the banquet in the Main Arena, Monday night. At the Production Clinic on Tuesday morning, speakers will discuss recommendations for sound broiler financing, a
new approach to interior egg quality problems and the latest trends in the production of "family size" turkeys. The final session on Tuesday afternoon will be devoted to a discussion of the affect of the defense on the poultry and egg industry and a discussion of the 1951 outlook for egg production and consumption. NATIONAL EGG PRODUCTS ASSOCIATION
The National Egg Products Association will hold its Annual Meeting in conjunction with the Institute of American Poultry Industries at the Fact Finding Conference in Kansas City, Missouri, on Sun-
(Continued on page 142)
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spot test is conducted indicates that it may be useful as a finding test in pullorum disease detection, especially among turkeys where large numbers of serum samples must be collected. However, among both chickens and turkeys the test may find its main application where suspicious reactors are encountered. The application of the test in this respect as well as its efficiency in detecting paratyphoid infection in turkeys are under further investigation.
131
T
* Paper No. 2573 scientific journal series, Minnesota Agricultural Experiment Station; taken from a thesis to be submitted to the graduate school of the University of Minnesota in partial fulfillment of the requirements for the degree of doctor of philosophy. 1 The author gratefully acknowledges the assistance of Miss Florence Jones. The investigations reported in this paper were supported in part by a grant-in-aid from the International Baby Chick Association.
has at his disposal antigens prepared from the standard or variant strain alone as well as polyvalent antigen composed of a mixture of standard and variant strains. Since, in most cases, it has not been practical to employ a double testing program, the latter antigen has often found favor in those flocks where both standard and variant types of pullorum disease have been encountered. A second factor in the effective application of the pullorum test is the problem of suspicious or so-called "pin-point" reactors. In this category are included those birds whose blood gives a late, very fine agglutination on the rapid whole blood plate test and a fine, easily dispersed, often low-titered reaction on the macroscopic tube test. These reactions may be encountered in certain flocks from year to year or they may be demonstrable for only a short period of time. In any event, they present a definite problem, especially when they are encountered in large numbers. While it is recognized that several factors may be involved in these atypical reactions, studies conducted so far suggest that cross-reacting agglutinins for S. pullorum occur in low or occasionally high titer in the blood of such birds. Investigations by Garrard et al. (1946, 1947), Carpenter et al. (1947) and Burton and Garrard (1948), have indicated that subclinical infections with coliforms, micrococci (staphylococci), enterococci, and other organisms may initiate these suspicious reactions.
125
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
HERE are certain factors that tend to interfere with the effective application of the agglutination test employed for the diagnosis of pullorum disease in chickens and turkeys. As the testing program has decreased the incidence of infected flocks, two of these factors have become of special concern to those engaged in the control of pullorum disease. First, there is the recent recognition by Younie (1941), Edwards and Bruner (1946) and others that Salmonella pullorum is subject to antigenic variation, giving rise to so-called standard and variant types. While the variant type of pullorum disease has been detected in various parts of the United States and Canada, more extensive serological typing is necessary to determine the true extent of variant infection. Investigations in recent years have established that either type of the disease may escape detection if an antigen prepared from the opposite type is used in testing. This fact has prompted a reconsideration of the antigen problem and has resulted in the appearance of a variety of antigens on the market. The tester now
126
J. E.
WILLIAMS
The variant and polyvalent pullorum antigens seem to show a greater affinity for cross-reacting agglutinins t h a n the standard antigen. Therefore, the former antigens are usually employed only in those flocks where the variant type of pullorum disease has been encountered. In view of the problem, efforts have been made to eliminate suspicious reac-
employed in screening out questionable reactors. T h e test and the results of investigations of its application to the diagnosis of pullorum disease in chickens and turkeys are discussed in this paper. EQUIPMENT
The following equipment is required for conducting the test:
FIG. 1. 1. A positive spot test using polyvalent stained antigen. Note that the antigen has been fixed in the center of the drop. 2. A negative test using polyvalent antigen. The antigen is distributed throughout the drop. 3. A negative test with a suspicious serum sample using polyvalent antigen.
tors through the development of more specific antigens for testing. However, the problem has persisted, and large numbers of suspicious reactors have been submitted to culture with negative results. There is no reliable method available for screening out suspicious reactors with any degree of certainty. T h e use by Castaneda (1950) of a unique and simple spot test (surface fixation test) for the detection of brucellosis, prompted our investigation of the application of this test to the diagnosis of pullorum disease. I t was believed t h a t because of the nature of the test it might be
1 loop (4.5 mm. internal diameter, 20 gauge wire) 1 loop (2 mm. internal diameter, 24 gauge wire) Filter paper (Schleicher & Schuell, No. 589, White Ribbon, or an equivalent moderately retentive grade) 2 petri dish covers containing physiological saline Pullorum stained antigen (4% cells) Serum samples to be tested PROCEDURE 1. Place a sheet of filter paper on top of a wire test tube rack. Mark the filter paper with the sample number or indicate in some other way the order of the samples to be tested. 2. Carefully place a drop of the serum on the filter paper using the 4.5 mm. loop. The loop should not be bulging with the serum but
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
4t|
SPOT TEST IN PULLORUM DIAGNOSIS
In conducting the test only one serum sample should be tested at a time and should be deposited on the filter paper immediately before the test is to be conducted. The loop should be rinsed in saline between each sample tested. Hemolyzed or partially jelled samples are unsuitable for testing. It is essential that steps 2, 3, and 4 be conducted in rapid succession. The antigen used is a doubly concentrated preparation of the usual crystal violet stained antigen prepared according to the specifications of the U. S. Bureau of Animal Industry, and employed in conducting the rapid whole blood test for pullorum disease. An antigen stained with aqueous hematoxylin proved inferior to the crystal violet antigen. The mechanism of the spot test seems to be a simple fixation of the antigen in the center of the drop of serum, due to a rapid and strong union of antigen and antibody, in the case of a positive serum sample. With a negative serum sample no such union takes place and the stained organisms are free to spread out through the area when the saline is added. Partial or weakly reacting agglutinins encountered in suspicious reactors are, in most cases, not capable of combining with the
127
antigen particles, in so short a period of time, or combine so loosely that the antigen particles are readily washed away with the saline (Figure 1; 3). RESULTS
At the time when this test came to our attention a group of twenty-two adult female chickens were being studied for agglutinin titers with pullorum antigens. These birds were accumulated from field flocks in which suspicious reactors had been encountered when the birds were tested with polyvalent pullorum antigen, using the whole blood plate test. The birds had been under observation for a period of eight months and had been tested at frequent intervals during this time, using a variety of plate and tube antigens. Sixteen of the birds were classified as suspicious reactors. Only two of the chickens were believed to be infected with pullorum disease as revealed by hightitered, coarse agglutination reactions and positive egg culture. Four of the birds were entirely negative with all the antigens that were used. When the serums of these birds were tested with the spot test using polyvalent pullorum antigen only two samples revealed positive tests and these were from the two birds that had previously been classified as infected. The organs of each individual bird were submitted to careful bacteriological examination using several different mediums in an effort to recover any organisms which they might harbor. S. pullorum was not isolated from any of the chickens except the two that revealed the positive reactions as noted above. It is of interest to mention an Escherichia coli organism isolated from the liver of bird No. 3, a suspicious reactor with variant and polyvalent antigens. A crystal violet stained antigen was prepared from this organism and used in the spot test
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
should contain enough so that when it spreads it will cover an area about 12 mm. in diameter. 3. Using the 2 mm. loop immediately place 1 bulging loopful of the stained antigen on the center of the drop of serum. It is most essential that the antigen be slowly and carefully lowered onto the drop of serum while holding the loop almost parallel to the paper. 4. Immediately deposit two or three drops of saline on top of the antigen drop using the 2 mm. loop. The saline will spread at once through the antigen-serum mixture. 5. Allow the test to dry slightly and read. Positive: The antigen will remain in the center of the drop (Figure 1; 1). Negative: The antigen will spread out through the drop to color the whole area (Figure 1; 2, 3).
-
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#
:;:
TABLE 1.—Results of spot test with serum samples of thirty-two chickens
Agglutination
Spot test*
Culture results
No. of birds
Suspicious Suspicious Suspicious Suspicious Positive Negative Positive Positive Negative Positive Negative
Negative Negative Positive Positive Positive Negative Positive Negative Positive Negative Negative
Negative S. pullorum S. pullorum Negative S. pullorum Negative Negative S. pullorum S. pullorum Negative S. pullorum
14 0 0 1 10
s 2
0 0 0 0
* The readings noted were obtained with polyvalent stained antigen except for one infected bird that would have been classified as negative if standard antigen had not been used. Suspicious agglutination—Those samples revealing a slow, finely granular agglutination with the rapid whole blood plate test and/or a low or occasionally high-titered, easily dispersed reaction with incomplete supernatant clearing with the tube test. Positive agglutination—Rapid, coarse agglutination with the plate test and firm, coarse agglutination with clear supernatant at a dilution of 1-25 or higher with the tube test.
with the serum of bird No. 3. A positive reaction resulted from this combination; however, a negative test was obtained when a variant pullorum serum was tested using this antigen (Figure 2). These preliminary results indicated that the spot test might be useful in demonstrating the affinity of pullorum antigens for their homologous agglutinins, while eliminating any partial attraction for cross-reacting agglutinins. Serum samples from a group of thirtytwo chickens were subsequently tested. These chickens were selected at random from field flocks and included positive, negative, and suspicious reactors. After a period of observation, the birds were submitted to complete bacteriological examination for S. pullorum. Results obtained with the spot test on examining the serums of this group of chickens, are summarized in Table 1. It is
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
FIG. 2. 1. Positive spot test of the serum of a suspicious reactor (No. 3) using a stained antigen prepared from an E. coli culture isolated from the liver of the same bird. 2. Negative test of the serum of a variant infected chicken using the E. coli stained antigen.
SPOT TEST IN PULLORUM DIAGNOSIS
notable that almost one-half of the birds were suspicious reactors with the agglutination test. All of these birds gave negative readings on the spot test and did not yield S. pullorum on culture. The spot test detected every bird that gave a typical positive reaction with the agglutination test. S. pullorum was isolated from
V
alone had been used (Figure 3). All of the S. pullorum cultures isolated from these chickens were of the standard type. In conducting the spot tests on this group of birds the polyvalent antigen detected all of the positive birds except one. However, the serum of this bird gave a very markedly positive spot test with
SV
m I FIG. 3. 1, 2, 3. Tests of the serum of a standard infected chicken using standard (S), variant (V), and polyvalent (SV) stained antigens.
six of these birds. In no case were positive cultural results obtained with negative spot test results. Two birds were positive on all tests but did not yield S. pullorum. In conducting the agglutination tests noted in Table 1 both tube and plate antigens of standard, variant, and polyvalent types were used. Tube tests were incubated at 37 degrees C. for 24 hours. The results with these individual antigens have not been included because of the space that would be required. However, it should be pointed out that the majority of the suspicious reactions occurred with the variant and polyvalent antigens. Two of the positive birds would have been classified as negative if variant antigen
standard antigen while with variant antigen it was negative. Each year suspicious reactors are encountered in testing turkeys for pullorum disease. Here again these reactions are more frequently encountered with variant and polyvalent antigens. Spot test results correlated with agglutination and cultural results on a group of 104 turkeys are summarized in Table 2. Most of these turkeys were accumulated from various flocks as suspicious reactors during the 1949 testing season. At the time of bacteriological examination their serums were preserved with merthiolate and refrigerated. It is significant that approximately fifty
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 29, 2015
s
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percent of these birds while suspicious reactors with the agglutination test, were negative with the spot test and negative on culture. Three birds classified as suspicious agglutination reactors yielded S. pullorum on culture. One of these birds was positive with the spot test. Two of TABLE 2.—Results of spot test with serum samples of 104 turkeys Spot testf
Culture results
No. of birds
Suspicious Suspicious Suspicious Suspicious Suspicious Positive Negative Positive Positive Negative Positive Negative
Negative Negative Positive Positive Negative Positive Negative Positive Negative Positive Negative Negative
Negative S. pullorum S. pullorum Negative Paratyphoid S. pullorum Negative Negative S. pullorum S. pullorum Negative S. pullorum
50 2 1 5 2 9 28 4 0 0 2 1
* Tube tests were conducted using standard, variant, and polyvalent antigens. Tests were incubated at 37 degrees C. for 24 hours. t The results were obtained with polyvalent stained antigen. Suspicious agglutination—Indicated by a low or high-titered, fine, easily dispersed reaction with incomplete supernatant clearing on the tube test. Positive agglutination—Firm, coarse agglutination with clear supernatant at a dilution of 1-25 or higher with the tube test.
them would have been missed on the spot test. Five birds were suspicious agglutination reactors with positive spot tests and negative culture results. Two birds supicious with the 'agglutination test were negative on the spot test and yielded paratyphoid organisms on culture. Typing results on these paratyphoid cultures are not yet available. All of the S. pullorum cultures isolated from these turkeys were of the standard type. Fifty-five other turkey serum samples suspicious with the agglutination test were subsequently tested. The majority of these samples were negative with the spot test. Nine other birds revealing coarsely
DISCUSSION
The results presented above indicate that the spot test may serve as a useful adjunct to the agglutination test in the diagnosis of pullorum disease in chickens and turkeys. In testing typical pullorum reactors the sensitivity of the spot test correlated very closely with that of the agglutination test. The intensity of the fixation reaction in the spot test seems to be a function of the quantitative as well as the qualitative antibody content of the serum, and this may vary in degree depending upon the sample tested. Occasionally suspicious serum samples are encountered which reveal weakly positive spot test reactions. Such samples usually show an agglutination titer of 1-50 or even higher with pullorum antigens. Dilution of serum samples with saline tends to interfere to a certain extent with the mechanics of the test; however, it has been possible to show that undiluted samples typically positive with the tube agglutination test at a dilution of 1-25 give positive spot tests. In most cases, a polyvalent pullorum antigen used in the spot test to examine serums of known agglutinin content has proved equally effective in detecting either the standard or variant type of pullorum disease. However, in a few cases it was of advantage to use both the standard and variant antigens in order to demonstrate better the degree of reaction. In a number of instances an antigen prepared from the standard type of S. pullorum would not have detected a variant-inflected bird and vice-versa. The rapidity and ease with which the
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Agglutination*
positive tube agglutination tests were strongly positive spot test reactors. None of these birds have yet been submitted to bacteriological examination.
NEWS AND NOTES
SUMMARY The application of the spot test to the diagnosis of pullorum infection in chickens and turkeys is discussed. The method of conducting the test is presented. Results are cited which indicate that the test may be useful as an adjunct to the agglutination test, especially in flocks where suspicious reactors are a problem. REFERENCES Burton, W. H., and E. H. Garrard, 1948. Non-
pullorum agglutination reactions. IV. Reactions with pullorum antigen from fowl inoculated with coliform types. Can. J. Comp. Med. 12: 20-25. Carpenter, J. A., W. H. Burton and E. H. Garrard, 1947. Non-pullorum agglutination reactions. III. Reactions with pullorum antigen from fowl inoculated with an enterococcus. Can J. Comp. Med. 11:163-168. Castaneda, M. R., 1950. Surface fixation. A new method of detecting certain immunological reactions. Proc. Soc. Exp. Biol. Med. 73: 46-49. Edwards, P. R., and D. W. Bruner, 1946. Form variation in Salmonella pullorum and its relation to X strains. Cornell Vet. 36: 318-324. Garrard, E. H., L. A. McDermott, W. H . Burton and J. A. Carpenter, 1946. Non-specific pullorum agglutination reactions. I. Preliminary observations on fowl exhibiting non-specific reactions over an extended period. Can. J.'Comp. Med. 10: 342-347. Garrard, E. H., W. H. Burton, J. A. Carpenter and L. A. McDermott, 1947. Non-specific pullorum agglutination reactions. II. Post mortem studies on fowl exhibiting non-specific reactions over an extended period. Can. J. Comp. Med. 11: 102107. Younie, A. R., 1941. Fowl infection like pullorum disease. Can. J. Comp. Med. 5:164-167.
News and Notes {Continued from page 124)
egg products, and a close-up view of the Poultry and Egg National Board's retail training program. Food and Drug, and Public Health officials will participate in the Good Housekeeping Clinic on Monday afternoon when the Institute's new edition of Suggested Sanitary Standards for Poultry Plants will make its debut. Allan B. Kline, President of the American Farm Bureau Federation, will speak at the banquet in the Main Arena, Monday night. At the Production Clinic on Tuesday morning, speakers will discuss recommendations for sound broiler financing, a
new approach to interior egg quality problems and the latest trends in the production of "family size" turkeys. The final session on Tuesday afternoon will be devoted to a discussion of the affect of the defense on the poultry and egg industry and a discussion of the 1951 outlook for egg production and consumption. NATIONAL EGG PRODUCTS ASSOCIATION
The National Egg Products Association will hold its Annual Meeting in conjunction with the Institute of American Poultry Industries at the Fact Finding Conference in Kansas City, Missouri, on Sun-
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spot test is conducted indicates that it may be useful as a finding test in pullorum disease detection, especially among turkeys where large numbers of serum samples must be collected. However, among both chickens and turkeys the test may find its main application where suspicious reactors are encountered. The application of the test in this respect as well as its efficiency in detecting paratyphoid infection in turkeys are under further investigation.
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