- Email: [email protected]
Hemagglutinating Properties of Salmonella Typhimurium Strains Isolated from Avian Sources
848
T. J. SEXTON
in chicken sperm (Harris et ai, 1973; Sexton, 1973,1974). Obviously, one has to cast doubts on the merits of continuing to seek in vitro methods that would eliminate the contraceptive action of glycerol. ACKNOWLEDGEMENT The author wishes to express appreciation to T. A. Fewlass and H. A. Attick for their technical assistance. REFERENCES Harris, G. C , R. J. Thurston and J. Cundall, 1973.
Changes in the ultrastructure of the fowl spermatozoa due to rapid freeze-thaw. J. Reprod. Fert. 34: 389-394. Salisbury, G. W., and N. L. VanDemark, 1961. Physiology of Reproduction and Artificial Insemination of Cattle. W. H. Freeman & Co., San Francisco, Calif., pp. 384-399. Sexton, T. J., 1973. Effect of various cryoprotective agents on the viability and reproductive efficiency of chicken spermatozoa. Poultry Sci. 52: 1353-1357. Sexton, T. J., 1974. Comparison of various cryoprotective agents on washed chicken spermatozoa. 4. Metabolism and release of glutamicoxalacetic transaminase. Poultry Sci. 53: 284-287.
J. E . WILLIAMS AND A. D . WHITTEMORE
Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, Georgia 30601 (Received for publication September 3, 1974)
ABSTRACT A total of 565 strains of Salmonella typhimurium and S. typhimurium var. Copenhagen, isolated from avian species, were examined for their ability to cause hemagglutination of turkey erythrocytes. Results showed that 541 (95.7%) of the strains caused varying degrees of hemagglutination. The hemagglutinating principle was maintained in cultures stored at 5° C. as formalized suspensions for as long as 6 months. It was destroyed by exposure to a temperature of 80° C. for 1 minute or to absolute ethyl alcohol at 37° C. for 24 hours. The hemagglutinating principle was best preserved in cultures by infrequent transfers, growth in liquid media, lyophilization or storage at low temperatures. Addition of d-mannose to bacteria-erythrocyte mixtures at a final concentration of 0.5% completely inhibited the hemagglutinating activity of the cultures. A concentration of 1% d-mannose added to crystal-violet-stained antigen preparations of S. typhimurium eliminated entirely the hemagglutinating effect of positive cultures and did not interfere with regular agglutination reactions. The hemagglutinating activity of S. typhimurium strains is apparently due to the presence of rigid appendages (fimbriae) on the surface of the bacterial cells. Results from these studies showed that fimbriated cultures are quite common among strains of S. typhimurium. POULTRY SCIENCE 54: 848-855, 1975
INTRODUCTION HE ability of certain bacteria of the family Enterobacteriaceae to cause hemagglutination (HA) of the erythrocytes of avian as well as other animal species is ascribed to the presence on their surfaces of submicroscopic projections (fimbriae or pili) that act as organs of attachment (Duguid etai, 1955). The phenomenon is by no means limited to this family of organisms.
T
Fimbriation and resulting hemagglutination have been studied rather extensively; strains of Escherichia coli have been used (Guyot, 1908; Rosenthal, 1943; Kauffmann, 1948; Brinton et ai, 1954; Collier and de Miranda, 1955; Collier and Jacoeb, 1955; Collier et ai, 1955a, b; Duguid et ai, 1955; Maccacaro and Hayes, 1961; and Duguid, 1964). Considerably less attention has been given to the phenomenon as it exists naturally among
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Hemagglutinating Properties of Salmonella Typhimurium Strains Isolated from Avian Sources
SALMONELLA TYPHIMURIUM
Service's Center for Disease Control, Atlanta, Georgia, or the National Animal Disease Center, Ames, Iowa, where they had been serologically typed. The strains were maintained in sealed nutrient agar stabs kept in the dark at room temperature, and none had been transferred to fresh agar more than twice between the time they were received for typing and the time they were used in these studies. Many had been transferred only once. The occurrence of HA-positive and HA-negative strains among the cultures isolated from the various avian species that served as their source is presented in Table 1.
In the preparation of cell suspensions from solid media for HA studies, all strains were grown aerobically at 37° C. for 24 hours on slants of Trypticase-Soy agar (BBL) enriched with 0.1% cysteine mono-hydrochloride and 2.0% glycerine and with agar content adjusted to 3% (TSCG agar). The agar was dispensed in 5-ml. amounts into 16- x 150-mm. tubes MATERIALS AND METHODS with stainless steel slip-on closures and slantSelected for these studies were 565 strains ed to provide a long slant and very little butt. of S. typhimurium isolated from avian sources When the cells were harvested from the in the United States from 1952 through 1965. slants, 1.5 ml. of 0.85% saline containing 0.5% Of the 565 strains, 119 were classified as formalin was added aseptically to each tube the Copenhagen variety. All strains were pro- and the growth dislodged by rotation of the cured from either the U.S. Public Health tube on a Vortex Genie mixer. This procedure provided a cell suspension with a density approximately 100 times tube 1 of the McFarTABLE 1.—Hemagglutination activity o/S. typhimurium cultures isolated from avian species land scale (McFarland, 1907). Adjustments were made in the density of cell suspensions Total as required to provide the 100 x tube 1 no. of HA HA + cultures Species concentration.
Turkeys Chickens Pigeons Geese Ducks Canaries Sparrows Partridges Pheasants Parrots Sandgrouse Penguin Unidentified Total
289 202 26 8 6 4 3 3 3 2 1 1 17
279 198 24 8 4 3 0 3 2 2 1 0 17
565
541
10 4 2 0 2 1 3 0 1 0 0 1 0 24
In efforts to stimulate HA activity in negative cultures, selected strains were serially transferred at 24-hour intervals into 10 ml. amounts of fresh Brain Heart Infusion broth (Difco) and incubated aerobically at 37° C. After incubation, cultures were killed by the addition of a few drops of formalin, and the cells sedimented by centrifugation at 850 x gfor 30 minutes. The supernatant was poured off, and the sedimented cells were resus-
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salmonella cultures, specifically Salmonella typhimurium. Harris and Williams (1957) demonstrated hemagglutinating (HA) activity in 17 of 22 laboratory strains of S. typhimurium. Duguid and Gillies (1958) reported that 9 of 11 laboratory strains of S. typhimurium that they examined were HA positive when mixed with guinea pig, fowl, and horse erythrocytes. HA activity among strains of S. typhimurium has practical importance in that it has precluded the use of HA-positive cultures in the study of antigen preparations to be used with avian whole blood. The studies reported in this paper were undertaken to determine the extent of HA activity among strains of S. typhimurium isolated from various avian sources in the United States. During these studies, observations were also made regarding some of the fundamental properties of the HA phenomenon as exhibited by S. typhimurium.
849
850
J. E. WILLIAMS AND A. D. WHITTEMORE
In the HA tests, the macroscopic plate technique was used. Bacterial cell suspensions (100 x tube 1 of the McFarland scale) were used undiluted and in increasing serial two-fold dilutions that resulted in cell suspensions corresponding to McFarland densities ranging from 50 x tube 1 through 0.78 x tube 1. With a sterile Pasteur pipette, one drop of the bacterial cell suspension was deposited in the center of a 14-mm. ceramic ring on a chemically clean, glass slide [3 x 2 inches (7.7 x 5.4 cm.) with 12 ceramic rings per slide]. One drop of erythrocyte suspension was added and thoroughly mixed with the bacteria. The slides were placed on an automatic rotating machine set for 150 rotations per minute and rotated for 5 minutes. Hemagglutination was read macroscopically against a white background. Undiluted bacterial cell suspensions (100 x tube 1 of the McFarland scale) were exposed to chemical agents in the following manner to determine their effect on the HA properties of the organisms:
Ethyl Alcohol. Undiluted cell suspensions were sedimented by centrifugation, the supernatants discarded, and the cells reconstituted to their original volume (1 ml.) by the addition of absolute ethyl alcohol. The cell suspensions were placed at both 5° and 37° C. for 24 hours. After centrifugation, they were reconstituted to their original volume with 0.5% formalized saline and titrated for HA activity. d-Mannose. A concentrated aqueous solution of d-mannose was added to undiluted, high HA-titered cell suspensions in a quantity to provide a final concentration of 0.5% when the cells were mixed with erythrocytes in macroscopic plate tests. After addition of the d-mannose, the cell suspensions were tested for HA activity. A final concentration of 1% d-mannose was also added to experimental crystal-violetstained whole-blood S. typhimurium antigen prepared from a high titered, HA-positive strain of the organism. The antigen was tested against the whole blood of chickens immediately after addition of the d-mannose and at intervals up to 3 months. When not in use, the formalin-preserved antigen was stored at 5° C. A crystal-violet-stained antigen prepared from a known HA-negative strain of S. typhimurium was used as a control to confirm the HA-inhibitory effect of dmannose. Constant temperature water baths were used to determine the heat lability of the HA factor. Two-fold saline dilutions of each separate strain ranging in concentration from undiluted to 0.78 x tube 1, were prepared and exposed to temperatures of 50°, 60°, 70°, and 80° C. for 1 minute. After the exposure period, each concentration was tested for HA activity. The effect of storage temperatures on the HA activity of undiluted cell suspensions was determined under the following conditions: (a) Storage at 5° C. in 0.5% formalized saline
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pended in the small amount of liquid still in the tube. These suspensions were tested for HA activity in the same manner as those harvested from TSCG agar medium, as described below. Chicken erythrocytes were used in preliminary work. However, it was established early that turkey erythrocytes were considerably more sensitive than chicken erythrocytes to the HA effect; therefore, turkey erythrocytes were used in conducting all tests. Heparinized whole blood was collected from adult Beltsville White turkeys and washed three times in sterile 0.85% saline and resuspended to provide a 3% cell concentration. The erythrocytes were stored at 5° C. and fresh suspensions prepared as needed. More dense suspensions of red blood cells (30%) were also used in some of this work; however, tests with such dense suspensions were not as easily read as those in which 3% suspensions were used, and titers were not quite as high.
851
SALMONELLA TYPHIMURIUM
TABLE 2.—Hemagglutination titers of 20 representative strains ofS. typhimurium
lOOx
50 x
M 3 M 10 M233 M238 M243 M244 M252 M259 M273 M284 M298 M295 M302 M307 M339 M437 M483 M528 M539 M559
4 4 4 4 4 4 4 4 4 3 4 4 4 4 4 4 3 4 4 4
4 3 4 4 4 3 4 4 4 3 4 3 4 4 4 2 3 3 4 4
Concentration of bacterial cell suspensions*** 3.13x 25 x 12.5x 6.25x 4 2 4 4 2 1 4 4 3 3 4 2 4 3 4
3
2
2
+
-
-
3 2 1 +
4 4
-
2 3
+
-
1 2 4 3
3 2 4
-+ 1 3 2
3 1
+
-+
— —
-
2 1 3
-+ 2 1
* Times McFarland tube 1. **- = negative; 1 to 4 = increasing degrees of hemagglutination; for 6 months and (b) storage at -40° C. with periodic tests after thawing from the frozen state. RESULTS Of the 565 strains of S. typhimurium and S. typhimurium var. Copenhagen included in these studies, only 24 strains (4.3%) failed to cause hemagglutination of turkey erythrocytes. The other 541 strains (95.7%) caused varying degrees of hemagglutination, as shown for a representative group of 20 strains in Table 2. Titers varied from strains positive at a cell concentration no lower than 50 x tube 1 to those revealing some degree of hemagglutination at concentrations even as low as 0.78 x tube 1. No effort was made to correlate such variations with the degree of development of fimbriae on the bacterial cells. Duguid and Gillies (1958) reported that HA activity varied directly with the degree of fimbriation. Among a total of 119 S. typhimurium var. Copenhagen strains examined, only 2 (1.7%) were HA negative, whereas 22 (4.9%) of 446 strains of S. typhimurium examined were HA
2
-
2
0.78 x
+
-
3 3
1.56x
2 2 1
— 1
H-
2
— ± ±
trace of hemagglutination.
negative. No correlation could be drawn between the avian species or geographic location from which a specific culture was isolated and its HA properties. The smooth-rough variation status of these strains was not studied in detail; however, some of the strains did show obvious evidence of roughness. This property in a strain did not influence its ability to cause hemagglutination nor did it render HA-negative cultures positive. Ninety-three strongly HA-positive strains and 23 negative strains were randomly selected for more detailed studies. After storage of the 93 strongly positive strains in 0.5% formalized saline suspension at 5° C. for 6 months, we found that the cells retained most of their HA activity. The HA titers of the cells included in Table 2 after they had been stored for 6 months at 5° C. are presented in Table 3. Harris and Williams (1957) demonstrated that formalin has a preservative effect upon the HA properties of S. typhimurium. Cell suspensions frozen at -40° C. for 2 months retained essentially the same titers as those exhibited before freezing.
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Strain no.
852
J. E. WILLIAMS AND A. D. WHITTEMORE
TABLE 3.—Hemagglutination titers of the 20 representative strains ofS. typhimurium listed in Table 2 after storage at 5° C. for 6 months lOOx
50 x
M3 M 10 M233 M238 M243 M244 M252 M259 M273 M284 M289 M295 M302 M 307 M339 M437 M 483 M 528 M539 M559
4 4 4 4 3 3 4 4 4 3 4 4 4 4 4 3 3 4 4 4
4 3 4 3 1
Concentration of bacterial cell suspensions * 3.13x 25 x 12.5X 6.25x 2 3 1 +
2 1
+
-
-
4 4 1 3 4 1 3 2 4 1 2 3 4 3
0.78x
-
3 2
-t-
1.56x
2 2
3 3
-h
-
2 2
3 4 +
— 1
2 +
-
2
3 ±
—
-+-
1 2 1
2 3 2
Times McFarland tube 1. ** - = negative; 1 to 4 = increasing degrees of hemagglutination; ± = trace of hemagglutination. The effect of heat on the HA factor of four selected strains is shown in Table 4. The destructive effect of heat on the HA activity of 22 selected strains was evident first at 70° C. There was a marked decrease TABLE 4.-—Effect
Temperature and strains 60° C. M 18 M291 M 471 M551 70° C. M 18 M 291 M471 M551 75° C. M 18 M 291 M471 M551 80° C. M 18 M291 M471 M551
in the HA titer at 75° C. and a temperature of 80° C. for 1 minute completely destroyed the HA activity of all strains studied. Exposure of sedimented cells to absolute ethyl alcohol for 24 hours during storage at
of heat upon the hemagglutinating principle
Concentration of bacterial cell suspensions*** lOOx
50 x
25x
12.5x
6.25x
3.13x
1.56x
0.78 x
4 4 4 4 4 4 4 4 2 2 2 2
-
*Times McFarland tube 1. ** - = negative; 1 to 4 = increasing degrees of hemagglutination;
trace of hemagglutination.
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Strain no.
853
SALMONELLA TYPHIMURIUM TABLE 5
Strains
—Effect of absolute ethyl alcohol and d- mannose upon the hemagglutinating principle lOOx
50 x
Concentration of bacterial cell suspensions*'** 6.25x 3.13x 25x 12.5x
1.56x
0.78x
Untreated M 18 M252 M259 M260 Absolute ethyl alcohol (37° C) M 18 M 252 M 259 M 260
37° C. completely eliminated the HA properties of the cells (Table 5). Such a procedure may have application in the preparation of somatic fractions of S. typhimurium antigens that are to be used with avian whole blood. This treatment has the disadvantage of destroying flagellar antigenic components. When sedimented cells were treated with
trace of hemagglutination.
absolute alcohol and subsequently stored at 5° C. for 24 hours, there was a strong inhibitory effect upon the principle, but it was not entirely eliminated. Collier and de Miranda (1955) reported that the sugar, d-mannose, in a concentration as low as 0.0004% was capable of inhibiting the hemagglutination of avian cells by the HA
TABLE 6.—Hemagglutination-inhibitory effect of d-mannose in experimental crystal-violet-stained S. typhimurium rapid whole-blood antigen* WB antigen, HA + strain, d-mannose not added**'***
WB antigen, HA - strain, control** Bird no.
1
2
6 13 14 43 44 45 49 50 51 52 53 54 55 85 87
4 4 4 2 4 2 4 1 2 2 2 1
4 4 3 1 1 1 1 1 1 2 1 1 1
-
4
-+
3 4 4 3 -+-
+
+
1
-
1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
WB antigen, HA + strain, d- mannose added** 3
1
2
3
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
4 4 4 2 4
4 4 3
4 4 3
-1-
—
-
4 1 1 1 2 1
-
1
±
-
—
•+-
—
1
±
•+-
—
•+•
—
1 ±
± —
-
-
* 1 = 4/2/65; 2 = 5/17/65; 3 = 6/14/65. **- = negative; 1 to 4 = increasing degrees of agglutination; ± = trace of agglutination. ***Typical hemagglutination dominated all reactions observed with this antigen.
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d-mannose (0.5%) M 18 M 252 M 259 M 260 * Times McFarland tube 1. ** - = negative; 1 to 4 = increasing degrees of hemagglutination;
854
J. E. WILLIAMS AND A. D. WHITTEMORE
principle of E. coli. These findings were studied further and confirmed by Duguid and Gillies (1957), who used cultures of Shigella flexneri. In the present studies with S. typhimurium, a 0.5% concentration of d-mannose completely inhibited the HA properties of cultures that have a high degree of development of the principle (Table 5).
Duguid and Gillies (1957) reported that HA-negative strains of shigella could be ren-
DISCUSSION The results of this study confirm the frequent natural occurrence of HA properties in strains of S. typhimurium isolated from avian sources. A limited number of strains of S. typhimurium isolated from mammalian sources were also studied in the course of this work with results similar to those reported for avian strains. The HA principle is considered to be closely bound to the bacterial cell wall and to be due to the presence of protein macromolecules in the form of rigid appendages (fimbriae) on its surface (Brinton et al., 1961). The degree of HA activity was observed to vary widely among the strains examined. The environment to which cultures were exposed during growth had a marked effect upon their HA characteristics. The HA principle can be preserved best in cultures by infrequent stock transfers, growth in liquid media, lyophilization, or storage at low temperatures. The principle is heat labile. The demonstration that d-mannose can be incorporated into stained whole-blood S. typhimurium antigen to completely inhibit HA activity permits a much broader selection of strains to be used in studies of such antigens. One is not limited to the use of only naturally occurring HA-negative cultures, which would restrict considerably the selection of strains of S. typhimurium having superior antigenic qualities. No effort has been made to relate HA properties to other unique or distinct characteristics of the organisms, such as variations
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The inhibitory effect of d-mannose on the HA activity of strains was immediate and lasted indefinitely in cell suspensions maintained at 5° C. Addition of a drop of a concentrated aqueous solution of d-mannose to cells that have already undergone hemagglutination in the macroscopic plate test will immediately disperse the agglutinated cells. Hemagglutination of avian erythrocytes by experimental crystal-violet-stained S. typhimurium antigens for whole-blood testing, prepared from strongly HA-positive strains of the organism, is completely eliminated by the addition of a final concentration of 1% d-mannose to the antigen at the time of preparation (Table 6). A stained whole-blood antigen prepared from a HA-negative strain of S. typhimurium was used as a control in the tests reported in Table 6. The chickens tested for agglutinin response had been infected with S. typhimurium by subcutaneous inoculation when they were 1 day old. Presence of 1% d-mannose in the antigen has not interfered with regular agglutination tests. It maintained its HA-inhibitory effect in experimental stained whole-blood antigen kept at 5° C. for 3 months, which was the extent of these tests. Washing d-mannose treated, crystal-violet-stained S. typhimurium cells with several changes of saline revealed that such cells were again strongly HA positive. The HA-inhibitory effect of d-mannose for S. typhimurium is apparently not involved with a permanent change in the cells because it can be removed by washing.
dered positive by repeated serial passage in liquid broth culture. Twenty HA-negative strains of S. typhimurium were selected and transferred at 24-hour intervals to fresh tubes of Brain Heart Infusion broth (Difco) and incubated at 37° C. HA properties were tested at 5-day intervals up to day 40. Seven of the 20 cultures became HA positive during this period.
SALMONELLA
in virulence, antigenic qualities, or phage types. In view of the frequent occurrence of an HA principle in S. typhimurium cultures, further information is needed on the antigenic and serological properties of the principle itself. A better understanding of these properties could provide an improved means of detecting chronic carriers of the organisms in nature. REFERENCES
855
fische Hemmung der Coli-Haemagglutination. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 124-132. Duguid, J. P., 1964. Functional anatomy of Escherichia coli with special reference to enteropathogenic E. coli. Rev. Lat.-Amer. Microbiol. 7: 1-16. Duguid, J. P., and R. R. Gillies, 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Path. Bact. 74: 397-411. Duguid, J. P., and R. R. Gillies, 1958. Fimbriae and hemagglutinating activity in Salmonella, Klebsiella, Proteus, and Chromobacterium. J. Path. Bact. 75: 519-520. Duguid, J. P., I. W. Smith, G. Dempster and P. N. Edmunds, 1955. Nonflagellar filamentous appendages ("fimbriae") and hemagglutinating activity in Bacterium coli. J. Path. Bact. 70: 335-348. Guyot, G., 1908. Uber die Bakterille Hamagglutination. (Bakterio-Haemagglutination). Centralbl. f. Bakt. 47: 640-653. Harris, M. E., and J. E. Williams, 1957. The hemagglutinating properties of Salmonella typhimurium. Amer. J. Vet. Res. 18: 432-436. Kauffmann, F., 1948. On haemagglutination by Escherichia coli. Acta Path. Microbiol. Scand. 25: 502-506. Maccacaro, G. A., and W. Hayes, 1961. The genetics of fimbriation in Escherichia coli. Genet. Res. Camb. 2: 394-405. McFarland, J., 1907. The nephelometer: an instrument for estimating the numbers of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Amer. Med. Assoc. 49: 1176-1178. Rosenthal, L., 1943. Agglutinating properties of Escherichia coli. Agglutination of erythrocytes, leucocytes, thrombocytes, spermatozoa, spores of molds, and pollen by strains of E. coli. J. Bact. 45: 545-550.
NEWS AND NOTES (Continued from page 844)
as far as improving the quality of human foods is concerned. The consequences, instead, will be the expenditure of a great deal of money and effort to irritate agricultural producers unnecessarily, to decrease the efficiency of their operations, and to raise the price of their products to the consumer. We consider it important to avoid these consequences which, sooner or later, would become evident to all."
I.E.C. NOTES After many years of "unofficial" participation, Canada has now joined the International Egg Commission as a full member. Producers in Canada will be represented by a delegate from the Canadian Egg Producers' Council, while the trade will be represented by the Canadian Egg Marketing Agency. Council members will be appointed shortly.
(Continued on page 865)
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Brinton, C. C , A. Buzzell and M. A. Lauffer, 1954. Electrophoresis and phage susceptibility studies on a filament-producing variant of the E. coli B bacterium. Biochem. Biophys. Acta, 15: 533-542. Brinton, C. C , P. Gemski, S. Falkow and L. S. Baron, 1961. Location of the piliation factor on the chromosome of Escherichia coli. Biochem. Biophys. Res. Commun. 5: 293-298. Collier, W. A., and J. C. de Miranda, 1955. BacterienHaemagglutination. III. Die Hemmung der ColiHaemagglutination durch Mannose. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 133-140. Collier, W. A., and M. Jacoeb, 1955. BacterienHaemagglutination. I. Versuche mit einem Haemagglutinrenden Stamm von E. coli. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 113-123. Collier, W. A., V. A. Tiggelman-Van Krugten and T. T. A. Hung, 1955a. Bacterien-Haemagglutination. IV. Uber Haemagglutinations versuche bei 105 ColiStammen und Adsorption der Haemagglutinine. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 303-315. Collier, W. A., S. T. Wong and J. C. de Miranda, 1955b. Bacterien-Haemagglutination. II. Unspeci-
TYPHMURIUM
T. J. SEXTON
in chicken sperm (Harris et ai, 1973; Sexton, 1973,1974). Obviously, one has to cast doubts on the merits of continuing to seek in vitro methods that would eliminate the contraceptive action of glycerol. ACKNOWLEDGEMENT The author wishes to express appreciation to T. A. Fewlass and H. A. Attick for their technical assistance. REFERENCES Harris, G. C , R. J. Thurston and J. Cundall, 1973.
Changes in the ultrastructure of the fowl spermatozoa due to rapid freeze-thaw. J. Reprod. Fert. 34: 389-394. Salisbury, G. W., and N. L. VanDemark, 1961. Physiology of Reproduction and Artificial Insemination of Cattle. W. H. Freeman & Co., San Francisco, Calif., pp. 384-399. Sexton, T. J., 1973. Effect of various cryoprotective agents on the viability and reproductive efficiency of chicken spermatozoa. Poultry Sci. 52: 1353-1357. Sexton, T. J., 1974. Comparison of various cryoprotective agents on washed chicken spermatozoa. 4. Metabolism and release of glutamicoxalacetic transaminase. Poultry Sci. 53: 284-287.
J. E . WILLIAMS AND A. D . WHITTEMORE
Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, Georgia 30601 (Received for publication September 3, 1974)
ABSTRACT A total of 565 strains of Salmonella typhimurium and S. typhimurium var. Copenhagen, isolated from avian species, were examined for their ability to cause hemagglutination of turkey erythrocytes. Results showed that 541 (95.7%) of the strains caused varying degrees of hemagglutination. The hemagglutinating principle was maintained in cultures stored at 5° C. as formalized suspensions for as long as 6 months. It was destroyed by exposure to a temperature of 80° C. for 1 minute or to absolute ethyl alcohol at 37° C. for 24 hours. The hemagglutinating principle was best preserved in cultures by infrequent transfers, growth in liquid media, lyophilization or storage at low temperatures. Addition of d-mannose to bacteria-erythrocyte mixtures at a final concentration of 0.5% completely inhibited the hemagglutinating activity of the cultures. A concentration of 1% d-mannose added to crystal-violet-stained antigen preparations of S. typhimurium eliminated entirely the hemagglutinating effect of positive cultures and did not interfere with regular agglutination reactions. The hemagglutinating activity of S. typhimurium strains is apparently due to the presence of rigid appendages (fimbriae) on the surface of the bacterial cells. Results from these studies showed that fimbriated cultures are quite common among strains of S. typhimurium. POULTRY SCIENCE 54: 848-855, 1975
INTRODUCTION HE ability of certain bacteria of the family Enterobacteriaceae to cause hemagglutination (HA) of the erythrocytes of avian as well as other animal species is ascribed to the presence on their surfaces of submicroscopic projections (fimbriae or pili) that act as organs of attachment (Duguid etai, 1955). The phenomenon is by no means limited to this family of organisms.
T
Fimbriation and resulting hemagglutination have been studied rather extensively; strains of Escherichia coli have been used (Guyot, 1908; Rosenthal, 1943; Kauffmann, 1948; Brinton et ai, 1954; Collier and de Miranda, 1955; Collier and Jacoeb, 1955; Collier et ai, 1955a, b; Duguid et ai, 1955; Maccacaro and Hayes, 1961; and Duguid, 1964). Considerably less attention has been given to the phenomenon as it exists naturally among
Downloaded from http://ps.oxfordjournals.org/ at Monash University on May 4, 2015
Hemagglutinating Properties of Salmonella Typhimurium Strains Isolated from Avian Sources
SALMONELLA TYPHIMURIUM
Service's Center for Disease Control, Atlanta, Georgia, or the National Animal Disease Center, Ames, Iowa, where they had been serologically typed. The strains were maintained in sealed nutrient agar stabs kept in the dark at room temperature, and none had been transferred to fresh agar more than twice between the time they were received for typing and the time they were used in these studies. Many had been transferred only once. The occurrence of HA-positive and HA-negative strains among the cultures isolated from the various avian species that served as their source is presented in Table 1.
In the preparation of cell suspensions from solid media for HA studies, all strains were grown aerobically at 37° C. for 24 hours on slants of Trypticase-Soy agar (BBL) enriched with 0.1% cysteine mono-hydrochloride and 2.0% glycerine and with agar content adjusted to 3% (TSCG agar). The agar was dispensed in 5-ml. amounts into 16- x 150-mm. tubes MATERIALS AND METHODS with stainless steel slip-on closures and slantSelected for these studies were 565 strains ed to provide a long slant and very little butt. of S. typhimurium isolated from avian sources When the cells were harvested from the in the United States from 1952 through 1965. slants, 1.5 ml. of 0.85% saline containing 0.5% Of the 565 strains, 119 were classified as formalin was added aseptically to each tube the Copenhagen variety. All strains were pro- and the growth dislodged by rotation of the cured from either the U.S. Public Health tube on a Vortex Genie mixer. This procedure provided a cell suspension with a density approximately 100 times tube 1 of the McFarTABLE 1.—Hemagglutination activity o/S. typhimurium cultures isolated from avian species land scale (McFarland, 1907). Adjustments were made in the density of cell suspensions Total as required to provide the 100 x tube 1 no. of HA HA + cultures Species concentration.
Turkeys Chickens Pigeons Geese Ducks Canaries Sparrows Partridges Pheasants Parrots Sandgrouse Penguin Unidentified Total
289 202 26 8 6 4 3 3 3 2 1 1 17
279 198 24 8 4 3 0 3 2 2 1 0 17
565
541
10 4 2 0 2 1 3 0 1 0 0 1 0 24
In efforts to stimulate HA activity in negative cultures, selected strains were serially transferred at 24-hour intervals into 10 ml. amounts of fresh Brain Heart Infusion broth (Difco) and incubated aerobically at 37° C. After incubation, cultures were killed by the addition of a few drops of formalin, and the cells sedimented by centrifugation at 850 x gfor 30 minutes. The supernatant was poured off, and the sedimented cells were resus-
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salmonella cultures, specifically Salmonella typhimurium. Harris and Williams (1957) demonstrated hemagglutinating (HA) activity in 17 of 22 laboratory strains of S. typhimurium. Duguid and Gillies (1958) reported that 9 of 11 laboratory strains of S. typhimurium that they examined were HA positive when mixed with guinea pig, fowl, and horse erythrocytes. HA activity among strains of S. typhimurium has practical importance in that it has precluded the use of HA-positive cultures in the study of antigen preparations to be used with avian whole blood. The studies reported in this paper were undertaken to determine the extent of HA activity among strains of S. typhimurium isolated from various avian sources in the United States. During these studies, observations were also made regarding some of the fundamental properties of the HA phenomenon as exhibited by S. typhimurium.
849
850
J. E. WILLIAMS AND A. D. WHITTEMORE
In the HA tests, the macroscopic plate technique was used. Bacterial cell suspensions (100 x tube 1 of the McFarland scale) were used undiluted and in increasing serial two-fold dilutions that resulted in cell suspensions corresponding to McFarland densities ranging from 50 x tube 1 through 0.78 x tube 1. With a sterile Pasteur pipette, one drop of the bacterial cell suspension was deposited in the center of a 14-mm. ceramic ring on a chemically clean, glass slide [3 x 2 inches (7.7 x 5.4 cm.) with 12 ceramic rings per slide]. One drop of erythrocyte suspension was added and thoroughly mixed with the bacteria. The slides were placed on an automatic rotating machine set for 150 rotations per minute and rotated for 5 minutes. Hemagglutination was read macroscopically against a white background. Undiluted bacterial cell suspensions (100 x tube 1 of the McFarland scale) were exposed to chemical agents in the following manner to determine their effect on the HA properties of the organisms:
Ethyl Alcohol. Undiluted cell suspensions were sedimented by centrifugation, the supernatants discarded, and the cells reconstituted to their original volume (1 ml.) by the addition of absolute ethyl alcohol. The cell suspensions were placed at both 5° and 37° C. for 24 hours. After centrifugation, they were reconstituted to their original volume with 0.5% formalized saline and titrated for HA activity. d-Mannose. A concentrated aqueous solution of d-mannose was added to undiluted, high HA-titered cell suspensions in a quantity to provide a final concentration of 0.5% when the cells were mixed with erythrocytes in macroscopic plate tests. After addition of the d-mannose, the cell suspensions were tested for HA activity. A final concentration of 1% d-mannose was also added to experimental crystal-violetstained whole-blood S. typhimurium antigen prepared from a high titered, HA-positive strain of the organism. The antigen was tested against the whole blood of chickens immediately after addition of the d-mannose and at intervals up to 3 months. When not in use, the formalin-preserved antigen was stored at 5° C. A crystal-violet-stained antigen prepared from a known HA-negative strain of S. typhimurium was used as a control to confirm the HA-inhibitory effect of dmannose. Constant temperature water baths were used to determine the heat lability of the HA factor. Two-fold saline dilutions of each separate strain ranging in concentration from undiluted to 0.78 x tube 1, were prepared and exposed to temperatures of 50°, 60°, 70°, and 80° C. for 1 minute. After the exposure period, each concentration was tested for HA activity. The effect of storage temperatures on the HA activity of undiluted cell suspensions was determined under the following conditions: (a) Storage at 5° C. in 0.5% formalized saline
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pended in the small amount of liquid still in the tube. These suspensions were tested for HA activity in the same manner as those harvested from TSCG agar medium, as described below. Chicken erythrocytes were used in preliminary work. However, it was established early that turkey erythrocytes were considerably more sensitive than chicken erythrocytes to the HA effect; therefore, turkey erythrocytes were used in conducting all tests. Heparinized whole blood was collected from adult Beltsville White turkeys and washed three times in sterile 0.85% saline and resuspended to provide a 3% cell concentration. The erythrocytes were stored at 5° C. and fresh suspensions prepared as needed. More dense suspensions of red blood cells (30%) were also used in some of this work; however, tests with such dense suspensions were not as easily read as those in which 3% suspensions were used, and titers were not quite as high.
851
SALMONELLA TYPHIMURIUM
TABLE 2.—Hemagglutination titers of 20 representative strains ofS. typhimurium
lOOx
50 x
M 3 M 10 M233 M238 M243 M244 M252 M259 M273 M284 M298 M295 M302 M307 M339 M437 M483 M528 M539 M559
4 4 4 4 4 4 4 4 4 3 4 4 4 4 4 4 3 4 4 4
4 3 4 4 4 3 4 4 4 3 4 3 4 4 4 2 3 3 4 4
Concentration of bacterial cell suspensions*** 3.13x 25 x 12.5x 6.25x 4 2 4 4 2 1 4 4 3 3 4 2 4 3 4
3
2
2
+
-
-
3 2 1 +
4 4
-
2 3
+
-
1 2 4 3
3 2 4
-+ 1 3 2
3 1
+
-+
— —
-
2 1 3
-+ 2 1
* Times McFarland tube 1. **- = negative; 1 to 4 = increasing degrees of hemagglutination; for 6 months and (b) storage at -40° C. with periodic tests after thawing from the frozen state. RESULTS Of the 565 strains of S. typhimurium and S. typhimurium var. Copenhagen included in these studies, only 24 strains (4.3%) failed to cause hemagglutination of turkey erythrocytes. The other 541 strains (95.7%) caused varying degrees of hemagglutination, as shown for a representative group of 20 strains in Table 2. Titers varied from strains positive at a cell concentration no lower than 50 x tube 1 to those revealing some degree of hemagglutination at concentrations even as low as 0.78 x tube 1. No effort was made to correlate such variations with the degree of development of fimbriae on the bacterial cells. Duguid and Gillies (1958) reported that HA activity varied directly with the degree of fimbriation. Among a total of 119 S. typhimurium var. Copenhagen strains examined, only 2 (1.7%) were HA negative, whereas 22 (4.9%) of 446 strains of S. typhimurium examined were HA
2
-
2
0.78 x
+
-
3 3
1.56x
2 2 1
— 1
H-
2
— ± ±
trace of hemagglutination.
negative. No correlation could be drawn between the avian species or geographic location from which a specific culture was isolated and its HA properties. The smooth-rough variation status of these strains was not studied in detail; however, some of the strains did show obvious evidence of roughness. This property in a strain did not influence its ability to cause hemagglutination nor did it render HA-negative cultures positive. Ninety-three strongly HA-positive strains and 23 negative strains were randomly selected for more detailed studies. After storage of the 93 strongly positive strains in 0.5% formalized saline suspension at 5° C. for 6 months, we found that the cells retained most of their HA activity. The HA titers of the cells included in Table 2 after they had been stored for 6 months at 5° C. are presented in Table 3. Harris and Williams (1957) demonstrated that formalin has a preservative effect upon the HA properties of S. typhimurium. Cell suspensions frozen at -40° C. for 2 months retained essentially the same titers as those exhibited before freezing.
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Strain no.
852
J. E. WILLIAMS AND A. D. WHITTEMORE
TABLE 3.—Hemagglutination titers of the 20 representative strains ofS. typhimurium listed in Table 2 after storage at 5° C. for 6 months lOOx
50 x
M3 M 10 M233 M238 M243 M244 M252 M259 M273 M284 M289 M295 M302 M 307 M339 M437 M 483 M 528 M539 M559
4 4 4 4 3 3 4 4 4 3 4 4 4 4 4 3 3 4 4 4
4 3 4 3 1
Concentration of bacterial cell suspensions * 3.13x 25 x 12.5X 6.25x 2 3 1 +
2 1
+
-
-
4 4 1 3 4 1 3 2 4 1 2 3 4 3
0.78x
-
3 2
-t-
1.56x
2 2
3 3
-h
-
2 2
3 4 +
— 1
2 +
-
2
3 ±
—
-+-
1 2 1
2 3 2
Times McFarland tube 1. ** - = negative; 1 to 4 = increasing degrees of hemagglutination; ± = trace of hemagglutination. The effect of heat on the HA factor of four selected strains is shown in Table 4. The destructive effect of heat on the HA activity of 22 selected strains was evident first at 70° C. There was a marked decrease TABLE 4.-—Effect
Temperature and strains 60° C. M 18 M291 M 471 M551 70° C. M 18 M 291 M471 M551 75° C. M 18 M 291 M471 M551 80° C. M 18 M291 M471 M551
in the HA titer at 75° C. and a temperature of 80° C. for 1 minute completely destroyed the HA activity of all strains studied. Exposure of sedimented cells to absolute ethyl alcohol for 24 hours during storage at
of heat upon the hemagglutinating principle
Concentration of bacterial cell suspensions*** lOOx
50 x
25x
12.5x
6.25x
3.13x
1.56x
0.78 x
4 4 4 4 4 4 4 4 2 2 2 2
-
*Times McFarland tube 1. ** - = negative; 1 to 4 = increasing degrees of hemagglutination;
trace of hemagglutination.
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Strain no.
853
SALMONELLA TYPHIMURIUM TABLE 5
Strains
—Effect of absolute ethyl alcohol and d- mannose upon the hemagglutinating principle lOOx
50 x
Concentration of bacterial cell suspensions*'** 6.25x 3.13x 25x 12.5x
1.56x
0.78x
Untreated M 18 M252 M259 M260 Absolute ethyl alcohol (37° C) M 18 M 252 M 259 M 260
37° C. completely eliminated the HA properties of the cells (Table 5). Such a procedure may have application in the preparation of somatic fractions of S. typhimurium antigens that are to be used with avian whole blood. This treatment has the disadvantage of destroying flagellar antigenic components. When sedimented cells were treated with
trace of hemagglutination.
absolute alcohol and subsequently stored at 5° C. for 24 hours, there was a strong inhibitory effect upon the principle, but it was not entirely eliminated. Collier and de Miranda (1955) reported that the sugar, d-mannose, in a concentration as low as 0.0004% was capable of inhibiting the hemagglutination of avian cells by the HA
TABLE 6.—Hemagglutination-inhibitory effect of d-mannose in experimental crystal-violet-stained S. typhimurium rapid whole-blood antigen* WB antigen, HA + strain, d-mannose not added**'***
WB antigen, HA - strain, control** Bird no.
1
2
6 13 14 43 44 45 49 50 51 52 53 54 55 85 87
4 4 4 2 4 2 4 1 2 2 2 1
4 4 3 1 1 1 1 1 1 2 1 1 1
-
4
-+
3 4 4 3 -+-
+
+
1
-
1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
WB antigen, HA + strain, d- mannose added** 3
1
2
3
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
4 4 4 2 4
4 4 3
4 4 3
-1-
—
-
4 1 1 1 2 1
-
1
±
-
—
•+-
—
1
±
•+-
—
•+•
—
1 ±
± —
-
-
* 1 = 4/2/65; 2 = 5/17/65; 3 = 6/14/65. **- = negative; 1 to 4 = increasing degrees of agglutination; ± = trace of agglutination. ***Typical hemagglutination dominated all reactions observed with this antigen.
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d-mannose (0.5%) M 18 M 252 M 259 M 260 * Times McFarland tube 1. ** - = negative; 1 to 4 = increasing degrees of hemagglutination;
854
J. E. WILLIAMS AND A. D. WHITTEMORE
principle of E. coli. These findings were studied further and confirmed by Duguid and Gillies (1957), who used cultures of Shigella flexneri. In the present studies with S. typhimurium, a 0.5% concentration of d-mannose completely inhibited the HA properties of cultures that have a high degree of development of the principle (Table 5).
Duguid and Gillies (1957) reported that HA-negative strains of shigella could be ren-
DISCUSSION The results of this study confirm the frequent natural occurrence of HA properties in strains of S. typhimurium isolated from avian sources. A limited number of strains of S. typhimurium isolated from mammalian sources were also studied in the course of this work with results similar to those reported for avian strains. The HA principle is considered to be closely bound to the bacterial cell wall and to be due to the presence of protein macromolecules in the form of rigid appendages (fimbriae) on its surface (Brinton et al., 1961). The degree of HA activity was observed to vary widely among the strains examined. The environment to which cultures were exposed during growth had a marked effect upon their HA characteristics. The HA principle can be preserved best in cultures by infrequent stock transfers, growth in liquid media, lyophilization, or storage at low temperatures. The principle is heat labile. The demonstration that d-mannose can be incorporated into stained whole-blood S. typhimurium antigen to completely inhibit HA activity permits a much broader selection of strains to be used in studies of such antigens. One is not limited to the use of only naturally occurring HA-negative cultures, which would restrict considerably the selection of strains of S. typhimurium having superior antigenic qualities. No effort has been made to relate HA properties to other unique or distinct characteristics of the organisms, such as variations
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The inhibitory effect of d-mannose on the HA activity of strains was immediate and lasted indefinitely in cell suspensions maintained at 5° C. Addition of a drop of a concentrated aqueous solution of d-mannose to cells that have already undergone hemagglutination in the macroscopic plate test will immediately disperse the agglutinated cells. Hemagglutination of avian erythrocytes by experimental crystal-violet-stained S. typhimurium antigens for whole-blood testing, prepared from strongly HA-positive strains of the organism, is completely eliminated by the addition of a final concentration of 1% d-mannose to the antigen at the time of preparation (Table 6). A stained whole-blood antigen prepared from a HA-negative strain of S. typhimurium was used as a control in the tests reported in Table 6. The chickens tested for agglutinin response had been infected with S. typhimurium by subcutaneous inoculation when they were 1 day old. Presence of 1% d-mannose in the antigen has not interfered with regular agglutination tests. It maintained its HA-inhibitory effect in experimental stained whole-blood antigen kept at 5° C. for 3 months, which was the extent of these tests. Washing d-mannose treated, crystal-violet-stained S. typhimurium cells with several changes of saline revealed that such cells were again strongly HA positive. The HA-inhibitory effect of d-mannose for S. typhimurium is apparently not involved with a permanent change in the cells because it can be removed by washing.
dered positive by repeated serial passage in liquid broth culture. Twenty HA-negative strains of S. typhimurium were selected and transferred at 24-hour intervals to fresh tubes of Brain Heart Infusion broth (Difco) and incubated at 37° C. HA properties were tested at 5-day intervals up to day 40. Seven of the 20 cultures became HA positive during this period.
SALMONELLA
in virulence, antigenic qualities, or phage types. In view of the frequent occurrence of an HA principle in S. typhimurium cultures, further information is needed on the antigenic and serological properties of the principle itself. A better understanding of these properties could provide an improved means of detecting chronic carriers of the organisms in nature. REFERENCES
855
fische Hemmung der Coli-Haemagglutination. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 124-132. Duguid, J. P., 1964. Functional anatomy of Escherichia coli with special reference to enteropathogenic E. coli. Rev. Lat.-Amer. Microbiol. 7: 1-16. Duguid, J. P., and R. R. Gillies, 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Path. Bact. 74: 397-411. Duguid, J. P., and R. R. Gillies, 1958. Fimbriae and hemagglutinating activity in Salmonella, Klebsiella, Proteus, and Chromobacterium. J. Path. Bact. 75: 519-520. Duguid, J. P., I. W. Smith, G. Dempster and P. N. Edmunds, 1955. Nonflagellar filamentous appendages ("fimbriae") and hemagglutinating activity in Bacterium coli. J. Path. Bact. 70: 335-348. Guyot, G., 1908. Uber die Bakterille Hamagglutination. (Bakterio-Haemagglutination). Centralbl. f. Bakt. 47: 640-653. Harris, M. E., and J. E. Williams, 1957. The hemagglutinating properties of Salmonella typhimurium. Amer. J. Vet. Res. 18: 432-436. Kauffmann, F., 1948. On haemagglutination by Escherichia coli. Acta Path. Microbiol. Scand. 25: 502-506. Maccacaro, G. A., and W. Hayes, 1961. The genetics of fimbriation in Escherichia coli. Genet. Res. Camb. 2: 394-405. McFarland, J., 1907. The nephelometer: an instrument for estimating the numbers of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Amer. Med. Assoc. 49: 1176-1178. Rosenthal, L., 1943. Agglutinating properties of Escherichia coli. Agglutination of erythrocytes, leucocytes, thrombocytes, spermatozoa, spores of molds, and pollen by strains of E. coli. J. Bact. 45: 545-550.
NEWS AND NOTES (Continued from page 844)
as far as improving the quality of human foods is concerned. The consequences, instead, will be the expenditure of a great deal of money and effort to irritate agricultural producers unnecessarily, to decrease the efficiency of their operations, and to raise the price of their products to the consumer. We consider it important to avoid these consequences which, sooner or later, would become evident to all."
I.E.C. NOTES After many years of "unofficial" participation, Canada has now joined the International Egg Commission as a full member. Producers in Canada will be represented by a delegate from the Canadian Egg Producers' Council, while the trade will be represented by the Canadian Egg Marketing Agency. Council members will be appointed shortly.
(Continued on page 865)
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Brinton, C. C , A. Buzzell and M. A. Lauffer, 1954. Electrophoresis and phage susceptibility studies on a filament-producing variant of the E. coli B bacterium. Biochem. Biophys. Acta, 15: 533-542. Brinton, C. C , P. Gemski, S. Falkow and L. S. Baron, 1961. Location of the piliation factor on the chromosome of Escherichia coli. Biochem. Biophys. Res. Commun. 5: 293-298. Collier, W. A., and J. C. de Miranda, 1955. BacterienHaemagglutination. III. Die Hemmung der ColiHaemagglutination durch Mannose. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 133-140. Collier, W. A., and M. Jacoeb, 1955. BacterienHaemagglutination. I. Versuche mit einem Haemagglutinrenden Stamm von E. coli. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 113-123. Collier, W. A., V. A. Tiggelman-Van Krugten and T. T. A. Hung, 1955a. Bacterien-Haemagglutination. IV. Uber Haemagglutinations versuche bei 105 ColiStammen und Adsorption der Haemagglutinine. Antonie van Leeuwenhoek J. Microbiol. Serol. 21: 303-315. Collier, W. A., S. T. Wong and J. C. de Miranda, 1955b. Bacterien-Haemagglutination. II. Unspeci-
TYPHMURIUM