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Isolation of Salmonella Typhimurium from the Feces of Artificially Infected Poults1
Isolation of Salmonella Typhimurium from the Feces of Artificially Infected Poults1 H. C. GAUGER AND R. E. GREAVES
Department of Poultry Science, North Carolina State College of Agriculture and Engineering, Raleigh, N. C. (Received for publication July IS, 1946)
Typhimurium was recovered from the liver of each of 10 inoculated poults that died within 4 weeks after inoculation. Nine days after inoculation 12 surviving poults, 3 from each of the inoculated groups, and 3 control poults (contact controls) were placed together in an all-wire pen. Only one of the inoculated poults appeared sick. Their pen was cleaned and disinfected daily with 3 percent phenol. On days that the feces were to be culture the feet of the 12 inoculated and control birds were washed with 70 percent alcohol and each bird was placed in an individual, freshly cleaned and disinfected wire bottom cage. As soon as all birds had voided feces on clean paper under each cage they were returned to the group pen. Twenty-two days after inoculation the 12 inoculated and 3 contact control birds were each placed in separate wire-bottom cages.
N REVIEWING the literature on Salmonella typhimurium infection in turkeys the authors found no reports on routine culturing of feces from poults which survived S. typhimurium infection. It is known that S. typhimurium can be isolated from the feces of infected poults at autopsy, but investigations as to how long the organism is voided in the feces of poults that survive the disease have not been reported. Since infected feces may play a major role in the dissemination of S. typhimurium, as well as other Salmonella infections, it is the purpose of this paper to present data on the duration of the voiding of S. typhimurium in the feces of poults that survived artificial infection. MATERIALS AND METHODS
One hundred poults 5 days of age were divided into 5 groups of 20 each. The poults in 4 groups were inoculated with 1.0,0.5, 0.1, and 0.01 c c , respectively, of a 26-hour beef extract broth culture of S. typhimurium by putting the inoculum in empty crops with a pipette. The fifth group of 20 poults served as controls. Inoculated and control poults were brooded on hardware cloth in separate brooders. 5.
Fecal cultures were begun on the 10th day after inoculation and were made at irregular intervals thereafter. The minimum period of fecal culturing for the 12 inoculated and 3 contact control birds was 58 days and the maximum, 99 days. During the first 42 days of study only small intestinal voidings were cultured. Subsequent to the 42d day some cecal discharges were also cultured. Five of the 12 inoculated birds and 2 of
1 Contribution from the Department of Poultry Science, N. C. Ag. Expt. Sta. Published with the approval of the Director as paper No. 225 oi the Journal series.
4§
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015
I
S. TYPHIMURIUM IN FECES OF INFECTED POULTS
Technic of Culturing Tissues at Autopsy. The liver, gall-bladder, heart, lungs, pancreas, spleen, oviduct, ovary or testes, and kidneys were macerated in sterile mortars with sand and each macerated organ distributed to one or more large test tubes containing 25 cc. of tetrathionate broth prepared according to the formula of Jones (1936). Incubated tissue cultures were streaked out on plated Endo's medium and S. typhimurium-like colonies were picked, subcultured to agar slants, and identified. Mucosal scrapings from the duodenum, middle portion of the small intestine, and ceca were also cultured in tetrationate broth.
and gas production. Sucrose, lactose, and raffinose were not fermented. Lead acetate agar was blackened and acid produced in Jordon's tartrate agar. Indol was not formed. Cultures were titrated for H and O agglutinogens in type antisera and sample cultures were tested for their ability to absorb agglutinins from the type antisera. RESULTS
The number of S. typhimurium isolations and the duration of positive S. typhimurium feces are shown in the table. DISCUSSION An examination of the data in the table shows that S. typhimurium was isolated with great frequency from the feces of 11 of the 12 birds from the 10th day to the 23d day following artificial per os inoculation; subsequently, isolations were relatively infrequent. A reduction in the number of isolations started rather promptly after the 22d day after inoculation, at which time each bird was placed in an individual cage. It is not known whether individual pen isolation accounted for this reduction or whether a reduction in the number of S. typhimurium voided in the feces of survivors of infection naturally occurs during this period. In 10 of the inoculated birds whose feces were cultured for a period of 100 to 108 days after inoculation the last isolation of S. typhimurium was made on the 32, 37, 79, 96, 81, 67, 35, 49, 23, and 253 day after inoculation, respectively.
At autopsy 71 to 253 days after inoculation tissue cultures of 10 inoculated birds Identification of Organisms Recovered. and the 3 contact controls were negative Organisms recovered and recorded in the for S. typhimurium as were also the tissues table as 6". typhimurium were motile, gram of 12 of the 15 uninoculated control birds negative rods that fermented dextrose, which were autopsied when 2 to 8 months, xylose, maltose, rhamnose, trehalose, pf age. S. typhimurium was recovered arabinpse, and mannitol and caused acid from the cecal mucosa of 2 of the inocu*
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015
the 3 contact control birds were autopsied and their tissues cultured between the 61st and 97th day of fecal study. The remaining birds were autopsied and cultured within 5 months after fecal culturing was terminated. Feces from 15 control birds were also cultured from 1 to 5 times during the first 42 days of study. Technic of Culturing Feces. One-half to one gram of freshly voided feces from each bird was thoroughly broken up in 10 cc. of sterile water and the coarser particles allowed to settle out for 15 minutes. Using a sterile 1 cc. pipette, 1, 2, 3, and 4 drops of the fecal suspension from each specimen tube were added, respectively, to four tubes of tetrathionate broth as recommended by Jones (19361. After 24 hours incubation at 31°C, a 4 mm. loop'ful from each of the inoculated tubes was streaked to plated Endo's agar. The plates were incubated for 20 to 24 hours and 5. typhimurium-like colonies picked, subcultured to agar slants and identified
49
S
S
c
72 77
+
+
+ +
c
79
S C S C S
74
-
S
+
c
81
+
-
S :
c
96
CC contact controls C (by bird numbers) uninoculated controls C (in table) feces voided from ceca S feces voided from intestines ce. m. cecal mucosa
s S S S S S S S S S S S S S S S S S S Scs c +++++++++ ++ + + +++ + +++++ ++ ++++ + + + + + + _+ + + + + + + + + ++ +++++ + + + +++ ++ + + + ++++++++++ ++ + +++++++ ++++++++ ++++++++ - +- + + + ++ + + + 4- + + +
70
Days after Inoculation
10 11 12 14 16 17 21 23 25 28 30 32 35 37 39 42 44 46 49 51 67
Legend: R inoculated with 1.0 cc. of S. typhimurium W inoculated with 0.5 cc. of S. typhimurium B inoculated with 0.1 cc. of S. typhimurium N inoculated with 0.01 cc. of S. typhimurium
R4096 R4008 R4057 W4068 W4058 W4003 B4031 B4078 B4039 N4028 N4066 N4069 CC4089 CC4072 CC4100 C4062 C4074 C4092 C4059 C4088 C4063 C4017 C4065 C4080 C4070 C4083 C4071 C4093 C4082 C4081
Bird No.
:
(=
=
:
-
166 246 71 253 105 172 232 172 100 105 173 105 84 162 84 All but 4,074, 4,063, 4,071 autopsied when 2 to 8 months of age
negative
neg. neg. neg. neg. ce. m. neg. neg. neg. neg. neg. neg. ce. m. neg. neg. neg.
Autopsy S. days 98 100 102 106 108 after typhi, inocula- from S C s c s c S c s c tion
Recoveries of S. typhimurium/raw/eees of artificially infected poults beginning 10 days after inoculation
ownloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015 O
S. TYPmMURiuM I N F E C E S OF I N F E C T E D P O U L T S -
murium, feces from 12 poults were cultured on 33, 33, 21, 33, 31, 33, 33, 33, 30, 31, 33 and 22 days and S. typkimurium was recovered on 11, 7, 11, 12, 11, 12, 2, 12, 8, 8, 8, and 6 days, respectively, the last recovery being made on the 32,37, 39, 79, 96, 81, 67, 35, 49, 23, 23, and 67th day after inoculation, respectively. From the 10th to the 23d day after inoculation isolations of S. typkimurium were frequent. Subsequent to this period isolations were relatively infrequent. At autopsy, 71 to 253 days after inoculation, 5. typkimurium was isolated from the cecal mucosa of 2 of the birds autopsied on the 105th day after inoculation. Ten of the 12 inoculated birds threw off the infection by the time they were 15 weeks of age.
From the 67th to the 108th day after inoculation, during which cecal discharges were also cultured, it will be noted that of the 8 isolations made during this period, 3 were from intestinal feces and 5 from cecal feces. Since fewer cecal than small intestinal feces cultures were made during this period, it is quite probable that had cecal fecal cultures been made throughout the entire study the number of S. typkimurium isolations would have been in excess of the number reported above.
Three uninoculated (contact control) poults which were in contact with the inoculated poults for 13 days prior to being individually caged also voided the organism in their feces. Their feces were cultured on 27, 33, and 27 days and S. typkimurium was recovered on 1> -6, and 1 day, respectively, the last isolation-being made from one bird on the 64th day after final exposure. S. typkimurium was not isolated from the feces of 15 uninoculated controls or from the tissues of 12 of these birds autopsied when 2 to 8 months of age.
SUMMARY
REFERENCE
During a 99-day period beginning on the 10th day after per os inoculation of 5-day-old poults with Salmonella typki-
Jones, E. R., 1936. The use of brilliant green-eosin agar and sodium tetrathionate broth for the isolation of organisms of the typhoid group. Jour. Path, and Bact. 42: 455-467.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015
lated birds (Nos. 4058 and 4069) autopsied on the 105th day after inoculation. It is quite probable that had we continued culturing the feces of these two birds we might have recovered the organism from their feces beyond the maximum isolation period of 96 days after inoculation as established in these studies. On the basis of bacteriological results on the feces and tissues, 10 of the 12 inoculated poults threw off the infection by the time they were 15 weeks of age. It should also be noted that the 3 uninoculated poults (contact controls), which were in contact with inoculated poults for 13 days prior to being caged individually, also voided S. typkimurium in their feces and in one instance (No. 4072) the organism was isolated from its feces on the 77th day or 64 days after final exposure.
51
Department of Poultry Science, North Carolina State College of Agriculture and Engineering, Raleigh, N. C. (Received for publication July IS, 1946)
Typhimurium was recovered from the liver of each of 10 inoculated poults that died within 4 weeks after inoculation. Nine days after inoculation 12 surviving poults, 3 from each of the inoculated groups, and 3 control poults (contact controls) were placed together in an all-wire pen. Only one of the inoculated poults appeared sick. Their pen was cleaned and disinfected daily with 3 percent phenol. On days that the feces were to be culture the feet of the 12 inoculated and control birds were washed with 70 percent alcohol and each bird was placed in an individual, freshly cleaned and disinfected wire bottom cage. As soon as all birds had voided feces on clean paper under each cage they were returned to the group pen. Twenty-two days after inoculation the 12 inoculated and 3 contact control birds were each placed in separate wire-bottom cages.
N REVIEWING the literature on Salmonella typhimurium infection in turkeys the authors found no reports on routine culturing of feces from poults which survived S. typhimurium infection. It is known that S. typhimurium can be isolated from the feces of infected poults at autopsy, but investigations as to how long the organism is voided in the feces of poults that survive the disease have not been reported. Since infected feces may play a major role in the dissemination of S. typhimurium, as well as other Salmonella infections, it is the purpose of this paper to present data on the duration of the voiding of S. typhimurium in the feces of poults that survived artificial infection. MATERIALS AND METHODS
One hundred poults 5 days of age were divided into 5 groups of 20 each. The poults in 4 groups were inoculated with 1.0,0.5, 0.1, and 0.01 c c , respectively, of a 26-hour beef extract broth culture of S. typhimurium by putting the inoculum in empty crops with a pipette. The fifth group of 20 poults served as controls. Inoculated and control poults were brooded on hardware cloth in separate brooders. 5.
Fecal cultures were begun on the 10th day after inoculation and were made at irregular intervals thereafter. The minimum period of fecal culturing for the 12 inoculated and 3 contact control birds was 58 days and the maximum, 99 days. During the first 42 days of study only small intestinal voidings were cultured. Subsequent to the 42d day some cecal discharges were also cultured. Five of the 12 inoculated birds and 2 of
1 Contribution from the Department of Poultry Science, N. C. Ag. Expt. Sta. Published with the approval of the Director as paper No. 225 oi the Journal series.
4§
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015
I
S. TYPHIMURIUM IN FECES OF INFECTED POULTS
Technic of Culturing Tissues at Autopsy. The liver, gall-bladder, heart, lungs, pancreas, spleen, oviduct, ovary or testes, and kidneys were macerated in sterile mortars with sand and each macerated organ distributed to one or more large test tubes containing 25 cc. of tetrathionate broth prepared according to the formula of Jones (1936). Incubated tissue cultures were streaked out on plated Endo's medium and S. typhimurium-like colonies were picked, subcultured to agar slants, and identified. Mucosal scrapings from the duodenum, middle portion of the small intestine, and ceca were also cultured in tetrationate broth.
and gas production. Sucrose, lactose, and raffinose were not fermented. Lead acetate agar was blackened and acid produced in Jordon's tartrate agar. Indol was not formed. Cultures were titrated for H and O agglutinogens in type antisera and sample cultures were tested for their ability to absorb agglutinins from the type antisera. RESULTS
The number of S. typhimurium isolations and the duration of positive S. typhimurium feces are shown in the table. DISCUSSION An examination of the data in the table shows that S. typhimurium was isolated with great frequency from the feces of 11 of the 12 birds from the 10th day to the 23d day following artificial per os inoculation; subsequently, isolations were relatively infrequent. A reduction in the number of isolations started rather promptly after the 22d day after inoculation, at which time each bird was placed in an individual cage. It is not known whether individual pen isolation accounted for this reduction or whether a reduction in the number of S. typhimurium voided in the feces of survivors of infection naturally occurs during this period. In 10 of the inoculated birds whose feces were cultured for a period of 100 to 108 days after inoculation the last isolation of S. typhimurium was made on the 32, 37, 79, 96, 81, 67, 35, 49, 23, and 253 day after inoculation, respectively.
At autopsy 71 to 253 days after inoculation tissue cultures of 10 inoculated birds Identification of Organisms Recovered. and the 3 contact controls were negative Organisms recovered and recorded in the for S. typhimurium as were also the tissues table as 6". typhimurium were motile, gram of 12 of the 15 uninoculated control birds negative rods that fermented dextrose, which were autopsied when 2 to 8 months, xylose, maltose, rhamnose, trehalose, pf age. S. typhimurium was recovered arabinpse, and mannitol and caused acid from the cecal mucosa of 2 of the inocu*
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015
the 3 contact control birds were autopsied and their tissues cultured between the 61st and 97th day of fecal study. The remaining birds were autopsied and cultured within 5 months after fecal culturing was terminated. Feces from 15 control birds were also cultured from 1 to 5 times during the first 42 days of study. Technic of Culturing Feces. One-half to one gram of freshly voided feces from each bird was thoroughly broken up in 10 cc. of sterile water and the coarser particles allowed to settle out for 15 minutes. Using a sterile 1 cc. pipette, 1, 2, 3, and 4 drops of the fecal suspension from each specimen tube were added, respectively, to four tubes of tetrathionate broth as recommended by Jones (19361. After 24 hours incubation at 31°C, a 4 mm. loop'ful from each of the inoculated tubes was streaked to plated Endo's agar. The plates were incubated for 20 to 24 hours and 5. typhimurium-like colonies picked, subcultured to agar slants and identified
49
S
S
c
72 77
+
+
+ +
c
79
S C S C S
74
-
S
+
c
81
+
-
S :
c
96
CC contact controls C (by bird numbers) uninoculated controls C (in table) feces voided from ceca S feces voided from intestines ce. m. cecal mucosa
s S S S S S S S S S S S S S S S S S S Scs c +++++++++ ++ + + +++ + +++++ ++ ++++ + + + + + + _+ + + + + + + + + ++ +++++ + + + +++ ++ + + + ++++++++++ ++ + +++++++ ++++++++ ++++++++ - +- + + + ++ + + + 4- + + +
70
Days after Inoculation
10 11 12 14 16 17 21 23 25 28 30 32 35 37 39 42 44 46 49 51 67
Legend: R inoculated with 1.0 cc. of S. typhimurium W inoculated with 0.5 cc. of S. typhimurium B inoculated with 0.1 cc. of S. typhimurium N inoculated with 0.01 cc. of S. typhimurium
R4096 R4008 R4057 W4068 W4058 W4003 B4031 B4078 B4039 N4028 N4066 N4069 CC4089 CC4072 CC4100 C4062 C4074 C4092 C4059 C4088 C4063 C4017 C4065 C4080 C4070 C4083 C4071 C4093 C4082 C4081
Bird No.
:
(=
=
:
-
166 246 71 253 105 172 232 172 100 105 173 105 84 162 84 All but 4,074, 4,063, 4,071 autopsied when 2 to 8 months of age
negative
neg. neg. neg. neg. ce. m. neg. neg. neg. neg. neg. neg. ce. m. neg. neg. neg.
Autopsy S. days 98 100 102 106 108 after typhi, inocula- from S C s c s c S c s c tion
Recoveries of S. typhimurium/raw/eees of artificially infected poults beginning 10 days after inoculation
ownloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015 O
S. TYPmMURiuM I N F E C E S OF I N F E C T E D P O U L T S -
murium, feces from 12 poults were cultured on 33, 33, 21, 33, 31, 33, 33, 33, 30, 31, 33 and 22 days and S. typkimurium was recovered on 11, 7, 11, 12, 11, 12, 2, 12, 8, 8, 8, and 6 days, respectively, the last recovery being made on the 32,37, 39, 79, 96, 81, 67, 35, 49, 23, 23, and 67th day after inoculation, respectively. From the 10th to the 23d day after inoculation isolations of S. typkimurium were frequent. Subsequent to this period isolations were relatively infrequent. At autopsy, 71 to 253 days after inoculation, 5. typkimurium was isolated from the cecal mucosa of 2 of the birds autopsied on the 105th day after inoculation. Ten of the 12 inoculated birds threw off the infection by the time they were 15 weeks of age.
From the 67th to the 108th day after inoculation, during which cecal discharges were also cultured, it will be noted that of the 8 isolations made during this period, 3 were from intestinal feces and 5 from cecal feces. Since fewer cecal than small intestinal feces cultures were made during this period, it is quite probable that had cecal fecal cultures been made throughout the entire study the number of S. typkimurium isolations would have been in excess of the number reported above.
Three uninoculated (contact control) poults which were in contact with the inoculated poults for 13 days prior to being individually caged also voided the organism in their feces. Their feces were cultured on 27, 33, and 27 days and S. typkimurium was recovered on 1> -6, and 1 day, respectively, the last isolation-being made from one bird on the 64th day after final exposure. S. typkimurium was not isolated from the feces of 15 uninoculated controls or from the tissues of 12 of these birds autopsied when 2 to 8 months of age.
SUMMARY
REFERENCE
During a 99-day period beginning on the 10th day after per os inoculation of 5-day-old poults with Salmonella typki-
Jones, E. R., 1936. The use of brilliant green-eosin agar and sodium tetrathionate broth for the isolation of organisms of the typhoid group. Jour. Path, and Bact. 42: 455-467.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 16, 2015
lated birds (Nos. 4058 and 4069) autopsied on the 105th day after inoculation. It is quite probable that had we continued culturing the feces of these two birds we might have recovered the organism from their feces beyond the maximum isolation period of 96 days after inoculation as established in these studies. On the basis of bacteriological results on the feces and tissues, 10 of the 12 inoculated poults threw off the infection by the time they were 15 weeks of age. It should also be noted that the 3 uninoculated poults (contact controls), which were in contact with inoculated poults for 13 days prior to being caged individually, also voided S. typkimurium in their feces and in one instance (No. 4072) the organism was isolated from its feces on the 77th day or 64 days after final exposure.
51