The Antigenic Variation of Salmonella Pullorum1

ANTIGENIC VARIATION OF S. PULLORUM

B12, injected at a 2 meg. level into eggs from vitamin Bi2-deficient hens, significantly increased hatchability and viability of chicks. The quantitative dietary requirement of vitamin B12 for satisfactory hatchability was found to be no more than 1 meg. per kg. of diet. The weight of mature birds was not affected by the vitamin B12 supplements in this experiment.

Carver, J. S., and J. McGinnis, 1950. Effect of an "animal protein factor" fermentation product on hatchability of chicken eggs. Poultry Sci. 29: 307-309. Lillie, R. J., M. W. Olsen and H. R. Bird, 1949. Role of vitamin Bi 2 in reproduction of poultry. Proc. Soc. Exp. Biol. Med. 72: 598-602. Lindstrom, R. G., C. F. Petersen, A. C. Wiese and P. R. Moore, 1949. Effect on hatchability of fish-

meal fed to hens deficient in the unidentified hatchability factor. Poultry Sci. 28: 552-555. Milligan, J. J., and G. F. Combs, 1950. Vitamin B i 2 requirements for hatchability, viability and growth. Poultry Sci. 29: 772. Peeler, H. T., R. F . Miller, C. W. Carlson, L. C. Norris and G. F. Heuser, 1951. Studies of the effect of vitamin B ] 2 on hatchability. Poultry Sci. 30: 11-20. Pensack, J. M., R. M. Bethke and D . C. Kennard, 1949. The effect of fish meal and extracts of fish meal on hatchability of hen's egg and growth of progeny. Poultry Sci. 28: 398-405. Skinner, J. L., J. H. Quisenberry and J. R. Couch, 1951. High efficiency and APF concentrates in the ration of the laying fowl. Poultry Sci. 30: 319-324. Snedecor, G. W., 1946. Statistical Methods. 4th Edition. The Iowa State College Press, Ames, Iowa. Yacowitz, H., R. F. Miller, L. C. Norris and G. F. Heuser, 1952. Vitamin B ] 2 studies with the hen. Poultry Sci. 31:89-94.

The Antigenic Variation of Salmonella Pullorum* A. J. Luzzio, L. D.

BUSHNELL AND

L. E.

ERWIN

Department of Bacteriology, Kansas State Agricultural Experiment Station, Manhattan (Received for publication March 26, 1952)

T

HIS investigation was undertaken to gain additional information on the characteristics associated with regular and variant strains of Salmonella pullorum, with major emphasis being placed on the antigenic relationship of parent strains of this organism carried on a stock culture medium and daughter colonies grown on various types of media. METHODS

Seventy cultures of 3". pullorum were selected for studying regular and variant tendencies. Each culture exhibited typical reactions for 5". pullorum, as specified by Bergey's Manual of Determina-

tive Bacteriology, sixth edition, and were in the "smooth" phase. Each of the 70 strains was transferred to Difco stock culture agar slants, containing 1.5 percent Bacto agar, and incubated for 48 hours at 37°C. At the end of this period antigens were prepared for each strain and suspended in 0.3 percent phenolized saline to give a McFarland nephelometer turbidity reading of tube 1. In order to avoid the removal of any of the antigenic components of S. pullorum (Bushnell, 1949), the cells were not washed prior to making up the antigens. An antiserum specific for the X I I 3 (regular) factor was prepared by inoculating a goat2 with Salmonella paratyphi A.

1

Contribution No. 261 Department of Bacteriology, Kansas State Agricultural Experiment Station. Manhattan, Kansas.

2 The goat and horse were both negative for nonspecific reaction.

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REFERENCES

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A. J. LTJZZIO, L. D. BUSHNELL AND L. E. ERWIN TABLE 1.—Method used in determining the antigenic types of the different antigens

Culture no. 42 36 64 54 68

r

,. t,™

type

Regular

Dilution Serum type 4+ — 4+ 4+ — 4+ 4+ 4+ 4-j4+

1:10

1:20

4+ — 4+ 4+ — 4+ 4+ 4+ 4+ 4+

4+ —• 4+ 4+ — 4+ 4+ 4+ 4+ 4+

1:40 4+ '— 4+ — — 4+ — 4+ — — •

1:80 1:160 1:320 1:640 1:1280 4+ — 4+ — — 4+ — 4+ — —

4+ — 4+ — — 4+ — 4+ — —

4+ — 4+ — — 4+ — 4+ — — •

— — — — — — — — — —

.— — — — — — — — — —

var. durazzo and absorbing the serum obtained with a cell suspension prepared from Salmonella reading. The procedure followed was essentially that of Wright and Edwards (1948). A variant serum was prepared by inoculating a horse2 with a strain of Proteus reported to be specific for the variant, or XII 2 , factor (Gwatkin, 1946). Each antigen was tested with both sera and the titers thus obtained are assumed to indicate the relative sensitivity of the various antigens to the XII 3 and XII2 factors. The data given in Table 1 illustrate the types of titration values obtained on the 70 cultures of S. pullorum with the two sera, and also the method used in classifying each strain as determined by its antigenic composition. In Table 2 the 52 strains giving a higher agglutination titer with regular absorbed serum than with Proteus anti-serum are designated as regular strains, the 14 strains giving the reverse titration are classed as variant strains, while the 4 strains giving equal titers with both sera constitute the group of intermediate strains.

on Difco dehydrated stock culture agar, and offspring of these organisms when grown on various solid culture media. The greater part of this work was concentrated on the study of 6 typical strains consisting of 2 regular (22 and 42), 2 variant (63 and 64), and 2 intermediate strains (67 and 68). Endo's medium, eosin methylene blue agar, bismuth sulphite agar, S. S. agar, MacConkey's agar, stock culture agar, and nutrient agar were used as selective media in this study. The first five were prepared from Difco dehydrated products as directed, except that an additional 7.5 gm. Difco agar was added to each liter of stock culture agar to assure a firm medium. Each strain was streaked onto the seven different media and incubated for 48 hours at 37°C. At the end of this period 25 colonies were picked from each plate and typed; thus, a total of 175 colonies from each strain were studied and typed by the slide agglutination method. With the exception of a few minor deviations the procedure followed for slide agglutination was the same as that used by Wright and Edwards (1948).

ISOLATION OF COLONY TYPES FROM VARIOUS MEDIA

Again the colonies which agglutinated more readily with regular-absorbed serum than with Proteus anti-serum were classed as regular; those which agglutinated more readily with Proteus anti-serum than with regular-absorbed serum were classed as

The following experiment was set up in an attempt to determine the antigenic relationship between the parent strains of S. pullorum which were being carried

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Regular-absorbed Proteus Regular Regular-absorbed Proteus Variant Regular-absorbed Proteus Variant Regular-absorbed Proteus Intermediate Regular-absorbed Proteus

1:5

ANTIGENIC VARIATION OF S. PULLORUM TABLE 2.—Highest dilution showing positive ag-

glutination in the titration of 70 S. pullorum antigens with regular-absorbed, and Proteus anti-sera Serum-types Culture no. Regularabsorbed Proteus Regu ar group 1 2 3* 4 •

5

160 320 120 160 640 640 320 160 320 160 320 160 320 80 320 80 320 160 320 640 640 320 640 160 320 80 320 80 320 160 160 160 160 160 640 320

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

5 20 0 10 20 5 20 10 20 5 20 20 10 5 20 5 20 20 40 20 20 0 40 20 20 40 20 5 10 10 40 20 10 10 10 80

37 38 39 40 41 42* 43 44 45 46 47 48 49 50 51 52

1:640 1 40 1:160 1 10 1:640 1 10 1:160 1 5 1:160 1 5 1:320 1 0 1:160 1 5 1:160 1 10 1:320 1 10 1:160 1 5 1:160 1 10 1:320 1 5 1 20 1:320 1:160 1 10 1:10 1 5 1:320 1 20 Variant group 53 1:5 1:320 54 1:20 1:320 55 1:10 1:320 56 1:5 1:160 57 1:10 1:320 58 1:20 1:160 59 1:20 1:160 60 1:5 1:80 1:20 1:160 61 62 1:5 1:160 63* 1:0 1:160 64* 1:0 1:320 65 1:40 1:80 66 1:20 1:40 Intermediate group 67* 1:20 1 "20 68* 1:20 1 20 69 1:20 1 20 70 1:20 1 20

* Strains studied intensively.

variant; while colonies agglutinating equally as well with both sera were typed as intermediate. The cells of each daughter colony were agglutinated, to some degree, by both the regular-absorbed and Proteus anti-sera, indicating that both antigenic factors, XII 3 and XII 2 , were present in all the colonies. The speed and completeness with which the organisms of each colony agglutinated in both sera bore a close relationship to the results obatined with the parent strain, as recorded in Table 2. Thus with very few exceptions a single parent strain did not give rise to daughter colonies of both the regular and variant forms. Hence, attempts to isolate and propagate pure regular and pure variant strains were not successful.

DISCUSSION

The antigenic titrations recorded in Table 2 show that 52 of the 70 strains of 5. pullorum gave a higher titer, and more rapid and complete agglutination, with regular-absorbed serum than with Proteus anti-serum, and that 3 of these 52 strains did not react with Proteus antiserum at all. Fourteen of the 70 strains gave a higher titer with Proteus antiserum than with regular-absorbed serum, two of which gave negative reactions with regular-absorbed serum; while 4 of the 70 strains gave equal titers with both types of sera. From these results, it would appear that stabilization in the pure regular form had occurred in cultures 3, 22, and 42 and in pure variant forms in cultures 63 and 64. However, when these cultures were streaked on the various selective media used in this study they gave rise to daughter colonies which were agglutinated by both sera, and hence would be classed as regular, variant, or intermediate, depending upon the dominant antigenic factor. Variation in the speed with which the cells from the colonies agglutinated in both sera indicated that the phenomenon of antigenic variation in S. pullorum is quantitative as well as qualitative. The results, obtained with colony types grown on various media, suggest that the environment may determine, in part, the amounts of factors X I I 3 and XII 2 present in a given strain of S. pullorum. In agreement with Wright (1947) it is believed that strains of 61. pullorum possessing the complete antigenic structure can be employed for the detection of both the regular and variant types of pullorum infection. For the most successful practical use, however, a strain or combination of strains which will agglutinate equally as well in both regular-absorbed and Proanti-sera, preferably with a titer of

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6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22* 23 24 25 26 27 28 29 30 31 32 33 34 35 36

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Serum types Culture • no. Regularabsorbed Proteus

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F. H. BIRD

at least 1:640 with each serum, would be highly desirable. The location of such a strain may be accomplished by chance, or possibly by growing cultures in an environment which will increase the concentration of the regular and variant factors.

up, and that all colonies were regular or variant depending upon the parent type. Evidence is presented, however, suggesting that the antigenic structure of S. pullorum can be influenced quantitatively by environmental factors.

CONCLUSION In the study of 70 strains of 5. pullorum it was found that there was a tendency for antigenic stabilization to occur with the retention of both the XII3 and XII2 factors by the organism. Daughtercolony-type studies on 7 media revealed that parent strains gave rise to daughter colonies complete in their antigenic make

Bushnell, L. D., 1949. Unpublished data. Gwatkin, R., 1946. IX. Studies in pullorum disease. Canadn. J. Comp. Med. 10: 254-267. Wright, M. L., 1947. Selection of suitable antigenic strains of S. pullorum by single colony isolation. Canadn. J. Comp. Med. 11: 68-73. Wright, M. L., and P. R. Edwards, 1948. The serologic differentiation of 5. pullorum. Amer. J. Vet. Res. 9: 386-388.

REFERENCES

F. H. BIRD Eastern States Farmers' Exchange, Incorporated, Westbrook Laboratory, RFD 3, Rockrille, Connecticut (Received for publication May 9, 1952)

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HE lysine requirement for growth of chicks and poults has been determined by Almquist and Mecchi (1942) and by Grau et al. (1946). The results of investigations into the lysine requirements of laying hens have been reported by Grau (1947) and by Ingram and coworkers (1950). Reports on the amino acid requirements of chickens in the later stages of growth have not appeared in the literature. It is generally assumed that when the protein content of the chick starter ration is decreased from 20 percent to a 16 percent developing ration, the amino acid requirements of the animal are decreased in the same ratio. Since experimental proof has not been reported to substantiate this assumption, a series of investigations have been undertaken to determine the amino acid requirements of

chickens in later stages of development. The first study in this series dealing with the lysine requirement of eight-week old Rhode Island Red chickens is reported in this paper. EXPERIMENTAL PROCEDURE

Rhode Island Red chicks were used in this study. They were removed from the incubators when one day old, vaccinated against Newcastle disease by the intranasal method and brooded in electrically heated metal battery brooders equipped with raised wire screen floors. Feed and water were supplied ad libitum throughout the pre-test and test periods. A commercial chick starting ration of the highefficiency type was fed for the first eight weeks. At the end of this period the chickens were banded and segregated into the experimental groups on the basis of weight

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The Lysine Requirement of Eight-Week Old Chickens