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A Note on Artificially Infected Fowl as Carriers of Salmonella
A Note on Artificially Infected Fowl as Carriers of Salmonella N. E. GIBBONS AND R. L. MOORE
Division of Applied Biology, National Research Council, Ottawa, Canada (Received for publication September 14, 1945)
T
HE finding of Salmonella types in Canadian dried egg powder (Gibbons and Moore, 1944) raised the question of their mode of entrance. According to Jungherr and Clancy (1939), Mailman et al. (1942), Darby and Stafseth (1942), Edwards and Bruner (1943) and Hinshaw et al. (1944), Salmonellae are widely distributed in fowl in the United States, and they are probably more common in Canada than is realized. The organisms may be transmitted from infected ovaries to the yolk, or from infected fecal matter to the shell or the egg contents. Although definite reference to the first mode of transfer has not been found in chickens (Bunyea, 1942), (except, of course, S. pullorum), Hinshaw and his co-workers (1942, 1943) have shown that this is possible in turkeys. The transmission through fecal matter is obvious and it has been shown by Schalm (1937) that the organisms can penetrate the shell of chicken eggs, and by Pomeroy and Fenstermacher (1941a) that they can penetrate into turkey eggs. Recently, Watt (1945) has isolated S. Montevideo from the contents of hens' eggs which were responsible for an outbreak on shipboard. The present study was undertaken to elicit some information on how long infected chickens carry the organisms and whether the organisms could be isolated from many of the eggs of infected birds. Issued as National Research Council Publication No. 1367.
MATERIAL AND METHODS
Six Barred Rocks, approximately 18 months old, five of them pullorum positive, were observed over a period of 37 days before making any tests. The birds were kept in exhibition cages with nesting boxes attached. Clean shavings were provided daily, or more often if necessary, to reduce fecal contamination. The eggs were collected at least twice a day. Fecal samples were usually collected in the morning. Both eggs and fecal samples were examined within a few hours of collection. Bacteriological examination of the shells was performed by washing the eggs in a sterile beaker with 100 ml. of tetrathionate broth, the surface being scrubbed with a sterile swab. The broth was then transferred to a flask and incubated overnight. The egg meats were removed aseptically by a technique used previously by the authors (1944) and enriched in tetrathionate broth for 24 hours. All isolations were made on Difco SS agar and suspected colonies checked against O-sera. The fecal samples were also enriched in tetrathionate broth, about 5 gm. being added to 50 ml. Where estimates of numbers were made, 10 gm. of feces were emulsified in 90 ml. of water or tetrathionate broth and ten tubes of each of at least three dilutions put up in tetrathionate broth. The most probable number (M.P.N.) of organisms per gram was then calculated from the number of tubes which were positive on SS agar. 115
I
116
N. E. GIBBONS AND R. L. MOORE
TABLE 1.—Days from each feeding on which S. bareilly and S. pullorum were isolated from feces and eggs • Feces
Eggs
2nd feeding 3rd feeding 1st feeding (5 days later) (13 days after 2nd) Bird 1 ml. aqueous 1 ml. 24 hr. susp. S. bareilly 1 ml. aqueous (1,000,000/ml.) susp. 5 . bareilly broth culture (10,000,000/ml.) 5 . bareilly
After 1st feeding
1 d to
2 4 5 6
neg. . neg. 2 neg.
2,4 2 (pullorum—4) neg. (pullorum—2) neg.
1-16,18 1-21, 23-30, 32-38 1-9,11-15, 17-19* 1-13, 15-21
•3
to
to
"2 to
to
neg.
neg. neg.
6
neg. neg.
8
6,8, 6 10 neg. neg.
neg. neg.
8
neg. neg.
9
neg. neg.
neg.
2 3
8
1
0 6
0 1
1
8
"3 6
After 3rd feeding
After 2nd feeding
1
0 20
7
* Bird killed on 19th day.
The cultures used for feeding purposes were isolated from dried egg powder and had been typed by Dr. P. R. Edwards, Lexington, Ky. Dr. Edwards also kindly typed representative isolations from the birds. RESULTS
During the first month 63 samples of feces from 6 birds were examined and 5. pullorum isolated from 7 (11.1 percent) (actually all from two birds). No other types of Salmonella were encountered during this time. It might also be stated here that on autopsy only S. pullorum was found in any of the untreated birds, and that during the first 55 days 5. pullorum was isolated from the meats of 6 of 108 eggs (5.5 percent). The results of feeding S. bareilly to four birds are summarized in Table 1. When aqueous suspensions were fed (1st and 2nd) the organisms were excreted for not more than 4 days. When a broth culture was fed the organisms were excreted fairly continuously for periods of 18 to 38 days. S. bareilly was isolated from the shells of three eggs laid by one bird, while excreting this organism. 5. pullorum was isolated from the meat of one of these eggs.
Estimates of the number of Salmonella excreted in the feces were not made at the beginning. The most probable number per gram on a number of days is shown in Table 2. The number is large at first but declines rapidly and is maintained at a fairly low level. There are occasional ..increases which may be due to sampling error or possibly reinfection. When Salmonellae were not found in dilutions of 1-10 and 1-100 the M.P.N, is given as < 1 TABLE 2.—Excretion of S. bareilly by birds after oral
administration of broth cultures (most probable number of organisms per gram) Days from feeding 7 8 . 9 10 11 13 14 15 16 17 18 19 20 21 34 36 39
Bird 2
4
— — — 600 — 2
— — 8,000 — — 180+
635 2 1 <1 <1* <1 <1 <1
28 35 30 13 2.5 6.5 18+ <1* 1,800 19 <1
6
5
—
10,000
— — — 40 64 <1* 30 <1 <1* <1*
19,500 450 1,000 150 9 <1 35 <1* 160 <1* 180+ 18+ <1*
* Salmonella isolated from larger samples,
t Killed.
117
ARTIFICIALLY INFECTED FOWL AS CARRIERS OF SALMONELLA
per gram. In such instances Salmonellae were at times found in the larger samples (5 gm. or more). While the above four birds were excreting S. bareilly, this organism was isolated on three occasions from the feces of another bird (1), although it had never received an experimental infection. This may have been a cross infection or an accident, although care was taken to avoid contamination of the actual fecal samples. In an attempt to determine whether an organism in a watery suspension from an agar culture was always eliminated rapidly, bird No. 4, after 7 days in which no Salmonella organisms were isolated, was fed 0.5 ml. of such a suspension of S. bareilly containing 220 million organisms per ml. Organisms were eliminated the next day but no estimates were made until the second when the M.P.N, was 7,300 per gm. The third day two samples were examined having 2,600 and 5,700 per gm. The fourth day 5. bareilly was isolated from the large sample of feces but only S. pullorum from the dilutions, the M.P.N, for the latter being 7,500 per gm. On the fifth day only 5. pullorum was found, the M.P.N, being greater than 18,000 per gm. When the bird was killed eight days later only S. pullorum was found. To obtain some data on where the organisms localized, one bird (5) was killed after excreting S. bareilly for 19 days. S. bareilly was isolated from only one caecum (Table 3), although 5. pullorum was found in most of the other organs. Two birds (1 and 3) were given 0.5 ml. of a 24 hour broth culture of S. bareilly orally, and both began to excrete the organism two days later. One bird (1) continued to do so until killed on the seventh day when S. bareilly was isolated from various parts of the intestinal tract, and S. pullorum from the other organs. Isolations were made from the excrement of
TABLE 3.—Distribution ofS. pullorum and S. bareilly in birds at autopsy Bird
Duodenum Intestine, upper Intestine, lower Ceca Liver Gall-bladder Spleen Ovaries Oviduct Ova
1
2*
3
5
B B B
._ —
B B B B B
+
— —
+ + + + +
+
—
+ —. — — + .— + — + —
— — B
+ + + + + +
6t — — — — — — — — —
B S- bareilly isolated. + S. pullorum isolated. * killed 26 days after feeding. f killed 29 days after feeding.
the other bird (3) on only the third and fourth days but when killed on the sixth day S. bareilly was found in the intestinal tract and liver. From this it would seem that S. bareilly localizes in the intestinal tract and in the few birds studied was carried for a short time only. Pomeroy and Fenstermacher (194lb) found that S. typhi-murium localized in the intestinal tract of turkeys but may be permanent. Later (1943) other Salmonella types were also found to localize in the intestine. SUMMARY
When aqueous suspensions of S. bareilly from agar were fed adult hens the organisms were eliminated from the intestinal tract in two to four days: when broth cultures were fed, they were excreted over periods of 18 to 38 days. In adult fowl the organisms apparently localize in the intestinal tract. I t is therefore quite possible that eggs may be contaminated through fecal matter. In these experiments S. bareilly was isolated from the shells of 3 of 37 eggs laid while this organism was being excreted. REFERENCES
Bunyea, H., 1942. Miscellaneous diseases of poultry. U.S.D.A. Yearbook of Agriculture, Washington, p. 994.
118
N. E. GIBBONS AND R. L. MOORE
Darby, C. W. and H. J. Stafseth, 1942. Salmonella infection common to man, animals and birds. Proc. 46th Ann. Meeting of U. S. Livestock San. Assoc, 189-201. Edwards, P. R. and D. W. Bruner, 1943. The occurrence and distribution of Salmonella types in the United States. J. Infect. Dis. 72: 58-67. Gibbons, N. E. and R. L. Moore, 1944. Dried whole egg powder XI. Occurrence and distribution of Salmonella organisms in Canadian egg powder. Can. Jour. Research, F, 22:48-57. Gibbons, N. E., R. L. Moore and C. 0. Fulton, 1944. Dried whole egg powder XV. Growth of Salmonella and other organisms in liquid and reconstituted egg.sCan. Jour. Research, F, 22:169-173. Hinshaw, W. R., 1943. Diseases of turkeys. California Agr. Expt. Sta. Bull. 613: 68-70. Hinshaw, W. R., E. McNeil, and Taylor, T. J., 1942. Four years progress in eradication of pullorum disease from turkey flocks. Proc. 46th Ann. Meeting U. S. Livestock San. Assoc: 224237. Hinshaw, W. R., E. McNeil and T. J. Taylor, 1944.
Avian salmonellosis. Amer. Jour. Hyg. 40: 264278. Jungherr, E. and C. F. Clancy, 1939. Serological types of Salmonella isolated from paratyphoid in chicks. Jour. Infec. Dis. 64: 1-17. Mailman, W. L., J. F. Ryff and E. Matthews, 1942. Studies in the Salmonella group. Method of isolation and pathogenicity of strains occurring in the intestines of chickens. Jour. Infect. Dis. 70: 253-262. Pomeroy, B. S. and R. Fenstermacher, 1941a. Amer. Jour. Vet. Research 2: 285-291. Pomeroy, B. and R. Fenstermacher, 1941b. Salmonella infection in breeding turkeys. Jour. Amer. Vet. Med. Ass. 99: 216-217. Pomeroy, B. and R. Fenstermacher, 1943. Salmonella infection of breeding turkeys. Amer. Jour. Vet. Research 4:199-208. Schalm, O., 1937. Study of a paratyphoid infection in chicks. Jour. Infect. Dis. 61:208-216. Watt, J., 1945. An outbreak of Salmonella infection in man from infected chicken eggs. U.S. Public Health Repts. 60:835-839.
Division of Applied Biology, National Research Council, Ottawa, Canada (Received for publication September 14, 1945)
T
HE finding of Salmonella types in Canadian dried egg powder (Gibbons and Moore, 1944) raised the question of their mode of entrance. According to Jungherr and Clancy (1939), Mailman et al. (1942), Darby and Stafseth (1942), Edwards and Bruner (1943) and Hinshaw et al. (1944), Salmonellae are widely distributed in fowl in the United States, and they are probably more common in Canada than is realized. The organisms may be transmitted from infected ovaries to the yolk, or from infected fecal matter to the shell or the egg contents. Although definite reference to the first mode of transfer has not been found in chickens (Bunyea, 1942), (except, of course, S. pullorum), Hinshaw and his co-workers (1942, 1943) have shown that this is possible in turkeys. The transmission through fecal matter is obvious and it has been shown by Schalm (1937) that the organisms can penetrate the shell of chicken eggs, and by Pomeroy and Fenstermacher (1941a) that they can penetrate into turkey eggs. Recently, Watt (1945) has isolated S. Montevideo from the contents of hens' eggs which were responsible for an outbreak on shipboard. The present study was undertaken to elicit some information on how long infected chickens carry the organisms and whether the organisms could be isolated from many of the eggs of infected birds. Issued as National Research Council Publication No. 1367.
MATERIAL AND METHODS
Six Barred Rocks, approximately 18 months old, five of them pullorum positive, were observed over a period of 37 days before making any tests. The birds were kept in exhibition cages with nesting boxes attached. Clean shavings were provided daily, or more often if necessary, to reduce fecal contamination. The eggs were collected at least twice a day. Fecal samples were usually collected in the morning. Both eggs and fecal samples were examined within a few hours of collection. Bacteriological examination of the shells was performed by washing the eggs in a sterile beaker with 100 ml. of tetrathionate broth, the surface being scrubbed with a sterile swab. The broth was then transferred to a flask and incubated overnight. The egg meats were removed aseptically by a technique used previously by the authors (1944) and enriched in tetrathionate broth for 24 hours. All isolations were made on Difco SS agar and suspected colonies checked against O-sera. The fecal samples were also enriched in tetrathionate broth, about 5 gm. being added to 50 ml. Where estimates of numbers were made, 10 gm. of feces were emulsified in 90 ml. of water or tetrathionate broth and ten tubes of each of at least three dilutions put up in tetrathionate broth. The most probable number (M.P.N.) of organisms per gram was then calculated from the number of tubes which were positive on SS agar. 115
I
116
N. E. GIBBONS AND R. L. MOORE
TABLE 1.—Days from each feeding on which S. bareilly and S. pullorum were isolated from feces and eggs • Feces
Eggs
2nd feeding 3rd feeding 1st feeding (5 days later) (13 days after 2nd) Bird 1 ml. aqueous 1 ml. 24 hr. susp. S. bareilly 1 ml. aqueous (1,000,000/ml.) susp. 5 . bareilly broth culture (10,000,000/ml.) 5 . bareilly
After 1st feeding
1 d to
2 4 5 6
neg. . neg. 2 neg.
2,4 2 (pullorum—4) neg. (pullorum—2) neg.
1-16,18 1-21, 23-30, 32-38 1-9,11-15, 17-19* 1-13, 15-21
•3
to
to
"2 to
to
neg.
neg. neg.
6
neg. neg.
8
6,8, 6 10 neg. neg.
neg. neg.
8
neg. neg.
9
neg. neg.
neg.
2 3
8
1
0 6
0 1
1
8
"3 6
After 3rd feeding
After 2nd feeding
1
0 20
7
* Bird killed on 19th day.
The cultures used for feeding purposes were isolated from dried egg powder and had been typed by Dr. P. R. Edwards, Lexington, Ky. Dr. Edwards also kindly typed representative isolations from the birds. RESULTS
During the first month 63 samples of feces from 6 birds were examined and 5. pullorum isolated from 7 (11.1 percent) (actually all from two birds). No other types of Salmonella were encountered during this time. It might also be stated here that on autopsy only S. pullorum was found in any of the untreated birds, and that during the first 55 days 5. pullorum was isolated from the meats of 6 of 108 eggs (5.5 percent). The results of feeding S. bareilly to four birds are summarized in Table 1. When aqueous suspensions were fed (1st and 2nd) the organisms were excreted for not more than 4 days. When a broth culture was fed the organisms were excreted fairly continuously for periods of 18 to 38 days. S. bareilly was isolated from the shells of three eggs laid by one bird, while excreting this organism. 5. pullorum was isolated from the meat of one of these eggs.
Estimates of the number of Salmonella excreted in the feces were not made at the beginning. The most probable number per gram on a number of days is shown in Table 2. The number is large at first but declines rapidly and is maintained at a fairly low level. There are occasional ..increases which may be due to sampling error or possibly reinfection. When Salmonellae were not found in dilutions of 1-10 and 1-100 the M.P.N, is given as < 1 TABLE 2.—Excretion of S. bareilly by birds after oral
administration of broth cultures (most probable number of organisms per gram) Days from feeding 7 8 . 9 10 11 13 14 15 16 17 18 19 20 21 34 36 39
Bird 2
4
— — — 600 — 2
— — 8,000 — — 180+
635 2 1 <1 <1* <1 <1 <1
28 35 30 13 2.5 6.5 18+ <1* 1,800 19 <1
6
5
—
10,000
— — — 40 64 <1* 30 <1 <1* <1*
19,500 450 1,000 150 9 <1 35 <1* 160 <1* 180+ 18+ <1*
* Salmonella isolated from larger samples,
t Killed.
117
ARTIFICIALLY INFECTED FOWL AS CARRIERS OF SALMONELLA
per gram. In such instances Salmonellae were at times found in the larger samples (5 gm. or more). While the above four birds were excreting S. bareilly, this organism was isolated on three occasions from the feces of another bird (1), although it had never received an experimental infection. This may have been a cross infection or an accident, although care was taken to avoid contamination of the actual fecal samples. In an attempt to determine whether an organism in a watery suspension from an agar culture was always eliminated rapidly, bird No. 4, after 7 days in which no Salmonella organisms were isolated, was fed 0.5 ml. of such a suspension of S. bareilly containing 220 million organisms per ml. Organisms were eliminated the next day but no estimates were made until the second when the M.P.N, was 7,300 per gm. The third day two samples were examined having 2,600 and 5,700 per gm. The fourth day 5. bareilly was isolated from the large sample of feces but only S. pullorum from the dilutions, the M.P.N, for the latter being 7,500 per gm. On the fifth day only 5. pullorum was found, the M.P.N, being greater than 18,000 per gm. When the bird was killed eight days later only S. pullorum was found. To obtain some data on where the organisms localized, one bird (5) was killed after excreting S. bareilly for 19 days. S. bareilly was isolated from only one caecum (Table 3), although 5. pullorum was found in most of the other organs. Two birds (1 and 3) were given 0.5 ml. of a 24 hour broth culture of S. bareilly orally, and both began to excrete the organism two days later. One bird (1) continued to do so until killed on the seventh day when S. bareilly was isolated from various parts of the intestinal tract, and S. pullorum from the other organs. Isolations were made from the excrement of
TABLE 3.—Distribution ofS. pullorum and S. bareilly in birds at autopsy Bird
Duodenum Intestine, upper Intestine, lower Ceca Liver Gall-bladder Spleen Ovaries Oviduct Ova
1
2*
3
5
B B B
._ —
B B B B B
+
— —
+ + + + +
+
—
+ —. — — + .— + — + —
— — B
+ + + + + +
6t — — — — — — — — —
B S- bareilly isolated. + S. pullorum isolated. * killed 26 days after feeding. f killed 29 days after feeding.
the other bird (3) on only the third and fourth days but when killed on the sixth day S. bareilly was found in the intestinal tract and liver. From this it would seem that S. bareilly localizes in the intestinal tract and in the few birds studied was carried for a short time only. Pomeroy and Fenstermacher (194lb) found that S. typhi-murium localized in the intestinal tract of turkeys but may be permanent. Later (1943) other Salmonella types were also found to localize in the intestine. SUMMARY
When aqueous suspensions of S. bareilly from agar were fed adult hens the organisms were eliminated from the intestinal tract in two to four days: when broth cultures were fed, they were excreted over periods of 18 to 38 days. In adult fowl the organisms apparently localize in the intestinal tract. I t is therefore quite possible that eggs may be contaminated through fecal matter. In these experiments S. bareilly was isolated from the shells of 3 of 37 eggs laid while this organism was being excreted. REFERENCES
Bunyea, H., 1942. Miscellaneous diseases of poultry. U.S.D.A. Yearbook of Agriculture, Washington, p. 994.
118
N. E. GIBBONS AND R. L. MOORE
Darby, C. W. and H. J. Stafseth, 1942. Salmonella infection common to man, animals and birds. Proc. 46th Ann. Meeting of U. S. Livestock San. Assoc, 189-201. Edwards, P. R. and D. W. Bruner, 1943. The occurrence and distribution of Salmonella types in the United States. J. Infect. Dis. 72: 58-67. Gibbons, N. E. and R. L. Moore, 1944. Dried whole egg powder XI. Occurrence and distribution of Salmonella organisms in Canadian egg powder. Can. Jour. Research, F, 22:48-57. Gibbons, N. E., R. L. Moore and C. 0. Fulton, 1944. Dried whole egg powder XV. Growth of Salmonella and other organisms in liquid and reconstituted egg.sCan. Jour. Research, F, 22:169-173. Hinshaw, W. R., 1943. Diseases of turkeys. California Agr. Expt. Sta. Bull. 613: 68-70. Hinshaw, W. R., E. McNeil, and Taylor, T. J., 1942. Four years progress in eradication of pullorum disease from turkey flocks. Proc. 46th Ann. Meeting U. S. Livestock San. Assoc: 224237. Hinshaw, W. R., E. McNeil and T. J. Taylor, 1944.
Avian salmonellosis. Amer. Jour. Hyg. 40: 264278. Jungherr, E. and C. F. Clancy, 1939. Serological types of Salmonella isolated from paratyphoid in chicks. Jour. Infec. Dis. 64: 1-17. Mailman, W. L., J. F. Ryff and E. Matthews, 1942. Studies in the Salmonella group. Method of isolation and pathogenicity of strains occurring in the intestines of chickens. Jour. Infect. Dis. 70: 253-262. Pomeroy, B. S. and R. Fenstermacher, 1941a. Amer. Jour. Vet. Research 2: 285-291. Pomeroy, B. and R. Fenstermacher, 1941b. Salmonella infection in breeding turkeys. Jour. Amer. Vet. Med. Ass. 99: 216-217. Pomeroy, B. and R. Fenstermacher, 1943. Salmonella infection of breeding turkeys. Amer. Jour. Vet. Research 4:199-208. Schalm, O., 1937. Study of a paratyphoid infection in chicks. Jour. Infect. Dis. 61:208-216. Watt, J., 1945. An outbreak of Salmonella infection in man from infected chicken eggs. U.S. Public Health Repts. 60:835-839.