Vol. 11, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, Mar. 1975, p. 588-591 Copyright © 1975 American Society for Microbiology
NOTES Calf Ligated Intestinal Segment Test to Detect Enterotoxigenic Escherichia colil L. L. MYERS,* F. S. NEWMAN, G. R. WARREN, J. E. CATLIN, AND C. K. ANDERSON Veterinary Research Laboratory, Montana State University, Bozeman, Montana 59715
Received for publication 3 September 1974
The ligated intestinal segment test in the young calf was utilized to establish the enterotoxigenicity of approximately 600 Escherichia coli isolates obtained from fecal specimens or intestinal contents of calves with diarrheal disease. One hundred and fifty isolates were routinely tested in a single calf. There was little problem with false-positive reactions. False-negative reactions normally occurred only if the isolate was tested in the posterior 3 m of the small intestine. The ligated small intestine of the calf was distended by E. coli isolates of bovine, porcine, and human origin. Use of the calf ligated intestinal segment test is recommended to determine the enterotoxigenicity of E. coli isolates of bovine origin.
During the past 3 years we have employed the ligated intestinal segment (LIS) test in the newborn calf as a tool to ascertain the enterotoxigenicity of Escherichia coli isolates obtained from fecal specimens or intestinal contents of young calves with diarrheal disease (scours) in Montana. E. coli isolates with ability to distend the LIS are designated as enterotoxigenic E. coli (ETEC). This is a report of our procedures and results with the LIS test in the calf and the potential utility of this system for determining the enterotoxigenicity of E. coli isolates. A modification of the calf LIS test reported earlier in six calves (3) was conducted with 25 colostrum-fed, male Holstein calves from 1 to 8 days of age. Calves 2 to 8 days of age were deprived of feed and water for 24 to 36 h prior to surgery. The surgical procedure was performed using antiseptic precautions. Calves were initially tranquilized (1 ml of promazine containing 50 mg of promazine hydrochloride per ml given intravenously) and then given a general anesthetic of 5 to 10 ml of sodium pentobarbital (1 g/ml) intravenously; the volume given depended upon the response of the calf. A local anesthetic (2% lidocaine hydrochloride) was infused along the proposed incision line. A 10-cm incision was made through the right paralumbar fossa and ligatures (0.04-mm veta-
fil suture material) were placed around the intestine 4 to 5 cm apart beginning 2 to 3 m cranial to the ileocecal junction and proceeding anteriorly. Segments were inoculated beginning at the anterior end of the ligated section of small intestine with 2 ml of 18-h heart infusion broth cultures of E. coli (109 or 1010 viable cells/ml) using 25-gauge disposable needles. Procedure used for isolation and storage of cultures are given elsewhere (2). ETEC strain B44 was inoculated into two or three different segments of most of the calves as a positive control. The first 19 calves were used for study of known ETEC strains of bovine and porcine origin, ETEC and non-ETEC (NETEC) of human origin, and for testing approximately 200 E. coli isolates obtained from calves with diarrheal disease during the 1972 and 1973 beef calving seasons. In these 19 calves, every other
'Journal Series no. 551 from Agricultural Experiment Station, Montana State Univ., Bozeman, Mont. 588
segment was inoculated and each isolate was tested in at least two calves. Isolates giving 0 or 1+ distention in both calves were considered non-enterotoxigenic. An isolate was considered enterotoxigenic if it gave 3 or 4+ distention in two calves. Isolates giving 2+ distention in one of the two calves were retested in one or two additional calves. In the last six calves (used to determine the enterotoxigenicity of 373 E. coli isolated from calves with diarrheal disease during the 1974 beef calving season), every segment was inoculated with E. coli and isolates that gave 0 to 11+ distention or 3 or 4+ distention
VOL. 11, 1975
NOTES
589
(with 0 or 1 + distention in adjacent segments) Veterans Administration Hospital, Sepulveda, in one calf were not retested. Isolates that gave Calif. The 15 tox+ human isolates studies were 2+ distention were retested in an additional 9190, 9192, 9195, 10407, 10409, 13634, 15697-3, calf. The last seven calves were each given 1,800 15734-7, 15862-2, 16095, 16160-2, 16932, 16354, ml of electrolyte solution (Hypolyte, Elanco 19123, and 19584. Tox- human isolates tested Products Co.) intravenously during the 2.5-h were 11352, 10405, 19943, 17747, 15833-1, surgical procedure. 15862-1, 16162-2, 15830-1, 15136, 16355, llllA, Calves were euthanatized 24 h postsurgery 16297, 15872-4, 14653, 15800-3, and 15870-3. and the test section of small intestine was Figure 1 shows results of the LIS test when removed intact. The mesentery was partially 123 different field isolates were inoculated into dissected away from the individual segments as consecutive segments. In later calves, isolates an aid in evaluation of distention. Degree of with unclassified antigens 1 to 6 (2) were not distention was determined for each segment placed in adjacent segments, thus avoiding with a rating of 4+ for maximum distention consecutively distended segments. Results of (when the segment was tightly stretched from the LIS test in the first 10 calves for all E. coli fluid pressure in the lumen) to 0 for a segment isolates that gave at least 2+ distention in one essentially devoid of fluid. A rating of 2+ was calf are presented in Table 1. There were no one-half maximum distention. Three or 4+ cases in which an isolate gave a positive reacdistention (with 0 to 1+ distention in adjacent tion (3+ or 4+ distention with 0 or 1+ distensegments) was considered positive, whereas 0 or tion in adjacent segments) in one calf that was 1+ distention (when the positive control gave 3 not repeatable. We also did not observe cases in or 4+ distention) was considered negative. Iso- which a field isolate gave 0 or 1+ distention in lates that gave 2+ distention were retested. the first calf and 3+ or 4+ distention in the The human E. coli isolates tested (15 tox+ second calf, although this did occur with ETEC and 16 tox- as previously determined by the strain 60/1577. Cases were rarely observed in adult rabbit ileal loop test) were supplied by which an isolate gave 2+ distention in more Doyle J. Evans, Infectious Disease Service, than one calf. Commonly, an isolate giving 2+
FIG. 1. Results of the LIS test in which 123 E. coli isolates were studied. The number of segments with 4+, 3+ and 2+ distention were 31, 5, and 6, respectively.
590
TABLE 1. Distentiona of the calf LIS after inoculation * with viable E. coli Calf no. E. coli isolates
Known ETEC obtained outside Montana B44 466A 3267-2 60/1577
- 2 1 2
3
340 263
4
5
-
61 7 81 9I 10
3 3 3 4 3 4 3 4 4 4 2 2 4 3 3 3 1 4 3 4 3 4
-
4042
Arnold
4 2
3
4 4 4 1 1 1
Field isolates of ETEC (1972 calving
season) 17364B 17368 17379A 17380A 17381D 17394 17395 17403 17419-1 17419-2 17435 17451
INFECT. IMMUN.
NOTES
4 4 4 4 3 2 3 4 3 2 2 4 2 3 3 3 2 3
4
4 4 4 4 4 4 4 4 4
See text for explanation of distention. Blank spaces indicate the isolate was not tested. Arnold, 340, and 263 are porcine isolates; all others are bovine isolates. a
because of the large number of' isolates obtained for study during the 1974 calving season. Since false-positive reactions were rarely observed, there is little chance of erroneously considering a NETEC to be enterotoxigenic. False-negative reactions may occur if the isolate is placed in the posterior 3 m of' small intestine, but these can largely be detected by retesting all isolates inoculated posterior to the last positive segment. Fifteen of the 25 calves survived the 24-h postoperative period and several of' the calves were able to stand 24 h after surgery. Nine of the other 10 calves lived 12 to 20 h after surgery. Results were considered valid if the positive control (strain B44) gave at least 3+ distention. Segments inoculated with ETEC in calves that lived about 12 h usually gave a 2+ or 3+ response, although an occasional 4+ response occurred. Of the seven calves given intravenous electrolyte solution during surgery, only one lived less than 20 h after surgery. An average of' 147 segments was established in each of these seven calves. The surgical procedure was conducted as rapidly as possible in an attempt to increase survival of' the calves. Attempts were unsuccessful to obtain a suitable clamp to replace the relatively slow procedure of ligating segments of' TABLE 2. Distention of the LIS of six different calves after inoculation with ETEC strain B44 No. of segments
Hours calf
in each calf
postaurgery
established distention in the first calf' gave greater distention in the second calf. Significant distention of' uninoculated segments after surgery as was reported earlier (4) was not observed. In an occasional calf', there was 2+ or 3+ distention in one or two sections of the small intestine due to fluid and/or gas accumulation at the time of' surgery that remained throughout the postsurgical period. Intestinal segments need not be established in these areas. ETEC strain B44 occasionally did not cause distention when placed in the posterior 2 to 3 m of' the small intestine (Table 2). This observation conf'irms earlier reports (3, 4) that the posterior portion of' the calf' small intestine is poorlv reactive. The enterotoxigenicity of E. coli isolates obtained during the 1974 calving season was often determined using only one inoculated segment in one calf. This was done because of' the reliability of' the test in the f'irst 19 calves and
lived
Segment no. inoculated with B44
(numbering
Distention
from anterior
end)
53
24
5 35
3 3
63
24
1 29 63
4 4 4
77
24
49 75
4 4
94
24
1 42 88
4 4 2
196
22
1
196 200
20
1 200
3 -
3 -
VOL. 11, 1975
NOTES
591
the intestine. Different types of general anes- enterotoxigenicity of E. coli isolates of bovine thetics including gas anesthetics may be tried to origin. find the one most suitable for the young calf. This research was performed as a contribution to Western The 15 tox+ and 16 tox- E. coli isolates of Regional Research Project W-112. human origin gave comparable results in the The cooperation of individuals in supplying strains of E. calf test as was previously shown for the rabbit coli is appreciated. (1). Fourteen of the 15 tox + strains (13634 being LITERATURE CITED the exception) gave 3+ or 4+ distention in the 1. Evans, D. G., D. J. Evans, Jr., and S. L. Gorbach. 1973. calf and 14 of the 16 tox- strains (15800-3 and Identification of enterotoxigenic Escherichia coli and 15870 being exceptions) did not distend the serum antitoxin activity by the vascular permeability intestine of the calf. Sensitivity of the calf' factor assay. Infect. Immun. 8:731-735. intestine to enterotoxins produced by human 2. Myers, L. L. 1975. Characterization of Escherichia coli obtained from newborn calves with diarrhea. Infect. isolates of ETEC may be usef'ul for some studies Immun. 11:493-496. of diarrheal disease in humans. 3. Smith, H. W., and S. Halls. 1967. Observations by the E. coli isolates from humans, pigs, and calves ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs, and caused distention of the LIS of the newborn rabbits. J. Pathol. Bacteriol. 93:499-529. calf. In addition, E. coli isolates from sheep 4. Tennant, B., D. Harrold, and M. Reina-Guerra. 1972. to distend the small were reported intestine of' Physiologic and metabolic factors in the pathogenesis of the calf (3). The LIS test in the young calf may neonatal enteric infections in calves. J. Am. Vet. Med. for be the preferred method Assoc. 161:993-1007. determining the
INFECTION AND IMMUNITY, Mar. 1975, p. 588-591 Copyright © 1975 American Society for Microbiology
NOTES Calf Ligated Intestinal Segment Test to Detect Enterotoxigenic Escherichia colil L. L. MYERS,* F. S. NEWMAN, G. R. WARREN, J. E. CATLIN, AND C. K. ANDERSON Veterinary Research Laboratory, Montana State University, Bozeman, Montana 59715
Received for publication 3 September 1974
The ligated intestinal segment test in the young calf was utilized to establish the enterotoxigenicity of approximately 600 Escherichia coli isolates obtained from fecal specimens or intestinal contents of calves with diarrheal disease. One hundred and fifty isolates were routinely tested in a single calf. There was little problem with false-positive reactions. False-negative reactions normally occurred only if the isolate was tested in the posterior 3 m of the small intestine. The ligated small intestine of the calf was distended by E. coli isolates of bovine, porcine, and human origin. Use of the calf ligated intestinal segment test is recommended to determine the enterotoxigenicity of E. coli isolates of bovine origin.
During the past 3 years we have employed the ligated intestinal segment (LIS) test in the newborn calf as a tool to ascertain the enterotoxigenicity of Escherichia coli isolates obtained from fecal specimens or intestinal contents of young calves with diarrheal disease (scours) in Montana. E. coli isolates with ability to distend the LIS are designated as enterotoxigenic E. coli (ETEC). This is a report of our procedures and results with the LIS test in the calf and the potential utility of this system for determining the enterotoxigenicity of E. coli isolates. A modification of the calf LIS test reported earlier in six calves (3) was conducted with 25 colostrum-fed, male Holstein calves from 1 to 8 days of age. Calves 2 to 8 days of age were deprived of feed and water for 24 to 36 h prior to surgery. The surgical procedure was performed using antiseptic precautions. Calves were initially tranquilized (1 ml of promazine containing 50 mg of promazine hydrochloride per ml given intravenously) and then given a general anesthetic of 5 to 10 ml of sodium pentobarbital (1 g/ml) intravenously; the volume given depended upon the response of the calf. A local anesthetic (2% lidocaine hydrochloride) was infused along the proposed incision line. A 10-cm incision was made through the right paralumbar fossa and ligatures (0.04-mm veta-
fil suture material) were placed around the intestine 4 to 5 cm apart beginning 2 to 3 m cranial to the ileocecal junction and proceeding anteriorly. Segments were inoculated beginning at the anterior end of the ligated section of small intestine with 2 ml of 18-h heart infusion broth cultures of E. coli (109 or 1010 viable cells/ml) using 25-gauge disposable needles. Procedure used for isolation and storage of cultures are given elsewhere (2). ETEC strain B44 was inoculated into two or three different segments of most of the calves as a positive control. The first 19 calves were used for study of known ETEC strains of bovine and porcine origin, ETEC and non-ETEC (NETEC) of human origin, and for testing approximately 200 E. coli isolates obtained from calves with diarrheal disease during the 1972 and 1973 beef calving seasons. In these 19 calves, every other
'Journal Series no. 551 from Agricultural Experiment Station, Montana State Univ., Bozeman, Mont. 588
segment was inoculated and each isolate was tested in at least two calves. Isolates giving 0 or 1+ distention in both calves were considered non-enterotoxigenic. An isolate was considered enterotoxigenic if it gave 3 or 4+ distention in two calves. Isolates giving 2+ distention in one of the two calves were retested in one or two additional calves. In the last six calves (used to determine the enterotoxigenicity of 373 E. coli isolated from calves with diarrheal disease during the 1974 beef calving season), every segment was inoculated with E. coli and isolates that gave 0 to 11+ distention or 3 or 4+ distention
VOL. 11, 1975
NOTES
589
(with 0 or 1 + distention in adjacent segments) Veterans Administration Hospital, Sepulveda, in one calf were not retested. Isolates that gave Calif. The 15 tox+ human isolates studies were 2+ distention were retested in an additional 9190, 9192, 9195, 10407, 10409, 13634, 15697-3, calf. The last seven calves were each given 1,800 15734-7, 15862-2, 16095, 16160-2, 16932, 16354, ml of electrolyte solution (Hypolyte, Elanco 19123, and 19584. Tox- human isolates tested Products Co.) intravenously during the 2.5-h were 11352, 10405, 19943, 17747, 15833-1, surgical procedure. 15862-1, 16162-2, 15830-1, 15136, 16355, llllA, Calves were euthanatized 24 h postsurgery 16297, 15872-4, 14653, 15800-3, and 15870-3. and the test section of small intestine was Figure 1 shows results of the LIS test when removed intact. The mesentery was partially 123 different field isolates were inoculated into dissected away from the individual segments as consecutive segments. In later calves, isolates an aid in evaluation of distention. Degree of with unclassified antigens 1 to 6 (2) were not distention was determined for each segment placed in adjacent segments, thus avoiding with a rating of 4+ for maximum distention consecutively distended segments. Results of (when the segment was tightly stretched from the LIS test in the first 10 calves for all E. coli fluid pressure in the lumen) to 0 for a segment isolates that gave at least 2+ distention in one essentially devoid of fluid. A rating of 2+ was calf are presented in Table 1. There were no one-half maximum distention. Three or 4+ cases in which an isolate gave a positive reacdistention (with 0 to 1+ distention in adjacent tion (3+ or 4+ distention with 0 or 1+ distensegments) was considered positive, whereas 0 or tion in adjacent segments) in one calf that was 1+ distention (when the positive control gave 3 not repeatable. We also did not observe cases in or 4+ distention) was considered negative. Iso- which a field isolate gave 0 or 1+ distention in lates that gave 2+ distention were retested. the first calf and 3+ or 4+ distention in the The human E. coli isolates tested (15 tox+ second calf, although this did occur with ETEC and 16 tox- as previously determined by the strain 60/1577. Cases were rarely observed in adult rabbit ileal loop test) were supplied by which an isolate gave 2+ distention in more Doyle J. Evans, Infectious Disease Service, than one calf. Commonly, an isolate giving 2+
FIG. 1. Results of the LIS test in which 123 E. coli isolates were studied. The number of segments with 4+, 3+ and 2+ distention were 31, 5, and 6, respectively.
590
TABLE 1. Distentiona of the calf LIS after inoculation * with viable E. coli Calf no. E. coli isolates
Known ETEC obtained outside Montana B44 466A 3267-2 60/1577
- 2 1 2
3
340 263
4
5
-
61 7 81 9I 10
3 3 3 4 3 4 3 4 4 4 2 2 4 3 3 3 1 4 3 4 3 4
-
4042
Arnold
4 2
3
4 4 4 1 1 1
Field isolates of ETEC (1972 calving
season) 17364B 17368 17379A 17380A 17381D 17394 17395 17403 17419-1 17419-2 17435 17451
INFECT. IMMUN.
NOTES
4 4 4 4 3 2 3 4 3 2 2 4 2 3 3 3 2 3
4
4 4 4 4 4 4 4 4 4
See text for explanation of distention. Blank spaces indicate the isolate was not tested. Arnold, 340, and 263 are porcine isolates; all others are bovine isolates. a
because of the large number of' isolates obtained for study during the 1974 calving season. Since false-positive reactions were rarely observed, there is little chance of erroneously considering a NETEC to be enterotoxigenic. False-negative reactions may occur if the isolate is placed in the posterior 3 m of' small intestine, but these can largely be detected by retesting all isolates inoculated posterior to the last positive segment. Fifteen of the 25 calves survived the 24-h postoperative period and several of' the calves were able to stand 24 h after surgery. Nine of the other 10 calves lived 12 to 20 h after surgery. Results were considered valid if the positive control (strain B44) gave at least 3+ distention. Segments inoculated with ETEC in calves that lived about 12 h usually gave a 2+ or 3+ response, although an occasional 4+ response occurred. Of the seven calves given intravenous electrolyte solution during surgery, only one lived less than 20 h after surgery. An average of' 147 segments was established in each of these seven calves. The surgical procedure was conducted as rapidly as possible in an attempt to increase survival of' the calves. Attempts were unsuccessful to obtain a suitable clamp to replace the relatively slow procedure of ligating segments of' TABLE 2. Distention of the LIS of six different calves after inoculation with ETEC strain B44 No. of segments
Hours calf
in each calf
postaurgery
established distention in the first calf' gave greater distention in the second calf. Significant distention of' uninoculated segments after surgery as was reported earlier (4) was not observed. In an occasional calf', there was 2+ or 3+ distention in one or two sections of the small intestine due to fluid and/or gas accumulation at the time of' surgery that remained throughout the postsurgical period. Intestinal segments need not be established in these areas. ETEC strain B44 occasionally did not cause distention when placed in the posterior 2 to 3 m of' the small intestine (Table 2). This observation conf'irms earlier reports (3, 4) that the posterior portion of' the calf' small intestine is poorlv reactive. The enterotoxigenicity of E. coli isolates obtained during the 1974 calving season was often determined using only one inoculated segment in one calf. This was done because of' the reliability of' the test in the f'irst 19 calves and
lived
Segment no. inoculated with B44
(numbering
Distention
from anterior
end)
53
24
5 35
3 3
63
24
1 29 63
4 4 4
77
24
49 75
4 4
94
24
1 42 88
4 4 2
196
22
1
196 200
20
1 200
3 -
3 -
VOL. 11, 1975
NOTES
591
the intestine. Different types of general anes- enterotoxigenicity of E. coli isolates of bovine thetics including gas anesthetics may be tried to origin. find the one most suitable for the young calf. This research was performed as a contribution to Western The 15 tox+ and 16 tox- E. coli isolates of Regional Research Project W-112. human origin gave comparable results in the The cooperation of individuals in supplying strains of E. calf test as was previously shown for the rabbit coli is appreciated. (1). Fourteen of the 15 tox + strains (13634 being LITERATURE CITED the exception) gave 3+ or 4+ distention in the 1. Evans, D. G., D. J. Evans, Jr., and S. L. Gorbach. 1973. calf and 14 of the 16 tox- strains (15800-3 and Identification of enterotoxigenic Escherichia coli and 15870 being exceptions) did not distend the serum antitoxin activity by the vascular permeability intestine of the calf. Sensitivity of the calf' factor assay. Infect. Immun. 8:731-735. intestine to enterotoxins produced by human 2. Myers, L. L. 1975. Characterization of Escherichia coli obtained from newborn calves with diarrhea. Infect. isolates of ETEC may be usef'ul for some studies Immun. 11:493-496. of diarrheal disease in humans. 3. Smith, H. W., and S. Halls. 1967. Observations by the E. coli isolates from humans, pigs, and calves ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs, and caused distention of the LIS of the newborn rabbits. J. Pathol. Bacteriol. 93:499-529. calf. In addition, E. coli isolates from sheep 4. Tennant, B., D. Harrold, and M. Reina-Guerra. 1972. to distend the small were reported intestine of' Physiologic and metabolic factors in the pathogenesis of the calf (3). The LIS test in the young calf may neonatal enteric infections in calves. J. Am. Vet. Med. for be the preferred method Assoc. 161:993-1007. determining the
