The Prevalence of Enterotoxigenic Escherichia coli in the Feces of Calves with Diarrhea G. Sivaswamy and C. L. Gyles* ABSTRACT A study was undertaken to determine the prevalence of enterotoxigenicity among Escherichia coli isolated from calves with diarrhea and from a control group of normal calves. The test organisms consisted of 200 E. coli recovered from scouring calves less than two weeks of age and 100 E. coli from normal calves. The enterotoxigenicity of the cultures was evaluated by three methods, namely, injection of ligated segments of piglet intestine, injection of ligated segments of calf intestine and oral inoculation of suckling mice. Live cutures of all the test organisms were used for the ligated intestine studies whereas sterile broth culture supernatants were used in the suckling mouse tests. Of the isolates from scouring calves, 36% were enterotoxigenic in the piglet intestine and 28% in the calf intestine. Amongst the isolates from normal calves, none was enterotoxigenic in the piglet intestine and one was enterotoxigenic in the calf test system. The ligated piglet intestine was considered unsuitable for determining the enterotoxigenicity of bovine E. coli, whereas the ligated calf intestine test was satisfactory and correlated completely with the suckling mouse test. The enterotoxigenic E. coli of bovine origin produced an enterotoxin that resembled the heat stable enterotoxin of typical porcine enteropathogenic E. coli.
que celui de 100 autres souches d'E. coli isolees d'un groupe de veaux temoins et normaux. Les auteurs utiliserent les trois m6thodes suivantes pour verifier l'enterotoxigenicite' des cultures: l'inoculation de segments d'intestin ligatures, chez des porcelets et des veaux, ainsi que l'injection orale 'a des souriceaux. Ils utiliserent des cultures vivantes de tous les colibacilles pour l'inoculation des segments d'intestin ligatures, tandis qu'ils employierent le surnageant sterile du bouillon des cultures pour l'injection or-ale 'a des souriceaux. Des souches isolees des veaux atteints de diarrhee, 36% s'avererent enterotoxinogenes dans l'intestin du porcelet et 28%, dans celui du veau. Aucune des souches isolees des veaux temoins ne s 'avera enterotoxinogiene dans l'intestin du porcelet; l'une d'elles s'avera cependant enterotoxinogene dans celui du veau. La methode des ligatures de l'intestin du porcelet sembla impropre at la verification du pouvoir enterotoxinogiene des souches bovines d'E. coli; par ailleurs, celle des ligatures de l'intestin du veau s'avera satisfaisante et corrobora parfaitement les resultats obtenus avec les souriceaux. Les E. coli enterotoxinogenes d'origine bovine produisirent une enterotoxine semblable a l'enterotoxine thermostable des souches d'E. coli enteropathogenes porcines typiques.
INTRODUCTION
RESUME Cette experience visait 'a determiner le pouvoir enterotoxinogZene de 200 souches d'Escherichia coli isolees de veaux ages de moins de deux semaines et atteints de diarrhee, ainsi
*Department of Veterinary Microbiology and Immunology, Ontario Veterinary College Univeraity of Guelph, Guelph, Ontario NIG 2W1. Submitted September 24, 1975.
Volume 40 -July, 1976
A wide variety of infectious and noninfectious agents have been implicated as causes of diarrhea in young calves (1, 3, 4, 9, 13, 17). Among the infectious agents, specific viruses have recently been recognized as etiological agents of calf diarrhea (1, 9, 10, 18) and the bacterium E. coli has for a long time been considered an important cause of calf scours (4, 13). The recognition that E. coti which cause diarrhea in calves are characterized by their production of enterotoxin (14) has paved the way for studies to evaluate the importance of E. coli among the various
241
agents implicated in calf diarrhea. Determination of the prevalence of enterotoxigenic E. coli in the feces of calves with diarrhea is an effective way of assessing the importance of E. coli in the calf diarrhea syndrome. In one study conducted in Great Britain, Smith and Halls (13) suggested that only a small percentage of cases of calf scours is attributable to E. coli. The objective of this study, therefore, was to determine the frequency of occurrence of enterotoxicity among E. coli isolates from the feces of scouring and normal calves. For this purpose, three test systems were used. Initially, the piglet gut loop test system was used since the ligated intestine of young piglets appeared to respond to enterotoxigenic E. coli of bovine origin (15). Subsequently, the cultures were tested in ligated intestine of calves. Finally, tests for in vitro enterotoxin production were conducted in piglet and calf gut loops and in suckling mice.
BROTH CULTURES
Broth cultures for gut loop tests were prepared by inoculating organisms into Tryptic Soy Broth (TSB) in 20 ml screw capped bottles, which were incubated at 37°C for 18 hours in a water bath at a rotary speed of 150 r.p.m. ENTEROTOXIN PREPARATION
Soft agar (SA) and broth supernatant (BS) preparations were as described by Stevens (16). CONCENTRATION OF ENTEROTOXIN PREPARATION
Concentration involved precipitation followed by extraction. The procedure for precipitation was similar to that described by Smith and Halls (14). GUT LooP TEST
MATERIALS AND METHODS
Piglets - The surgical procedure followed was similar to that described by Gyles and Barnum (7). For testing live ANIMALS cultures, 20 to 30 segments were made in each pig and the first segment was infed and colostrum hundred sixty One jected with normal saline. The segments in seven day old piglets and 13 five day old colostrum fed calves were used for ligated each pig were organized into seven segintestine tests. Four day old suckling mice ment test areas with a central positive (Connaught Medical Research Laboratory control. The volume of inoculum was 1.5 strain) were used in tests for enterotoxi- ml. At post mortem the results were read by qualitative and quantitative methods. city. Segments with fluid were positive and those without fluid were negative. For quantitative evaluation, the fluid volumes CULTURES per cm of intestine for positive segments Three hundred strains of E. coli, 200 in a test area were expressed as percentfrom scouring calves and 100 from normal ages compared with the value of the positive control. The percentage was called the ones, were isolated from the feces of calves under two weeks of age. The strains were reaction index (RI) and a mean reaction isolated over the period 1969 to 1974 and index (MRI) was calculated for each isostored on Trypticase Soy Agar (TSA) late. Each isolate was initially tested four slants. The isolates were subjected to bio- times. Two additional tests were conducted chemical tests to confirm their identity as for isolates found positive in one or two E. coli. Escherichia coli strains PS274 (4) of the initial four tests. For testing BS preparations, eight to and Y97 which had previously been shown segments were made in each piglet. twelve of calves (Gyles, 1973, to dilate gut loops unpublished), were used as positive con- Each BS preparation was tested a total trol organisms when testing live cultures of four times, twice in the unheated form of E. coli. Enterotoxin preparation from and twice after treatment at 65°C for 15 strain Y97 was used as a positive control minutes. The volume of inoculum was 8 ml. Heated and unheated forms of BS preparawhen testing enterotoxin preparations.
242
Can. J. comp. Med.
tions were tested in adjacent segments in the same pig. Calves - Each calf was starved for 12 to 24 hours prior to surgery. Anaesthesia was induced and maintained with halothane and a laparotomy was carried out aseptically through a paramedian incision on the right side. Silk ligatures which occluded the lumen were placed throughout the small intestine, beginning approximately one metre from the pylorus. Sixty to ninety segments were made in each calf and 2 ml broth culture was injected into each segment. Throughout surgery the calf was given intravenously between one and two litres of a balanced electrolyte solution (Proionate'). Segments of intestine were organized in series of 11 segment test areas with a central positive control. A negative control segment injected with TSB was included following every 22 segments. Three tests (each in a different calf) were conducted for each of the isolates classified enterotoxigenic in the piglet system. Isolates nonenterotoxigenic or doubtful in the piglet system were subjected initially to two tests but were subjected to an additional test when positive reactions were obtained. BS preparations were tested in the anterior 140-150 cm of the area of intestine selected for ligation. In each calf, 14 or 15 segments were prepared and injected with 8 ml of BS preparation. The seventh segment was injected with the negative control and the last segment of the series with the positive control. Each BS preparation was tested once in the unheated form and four preparations were also evaluated in paired tests of heated (65°C for 15 minutes) and unheated BS preparations. SUCKLING MOUSE TEST The procedure followed was similar to that described by Dean and co-workers (2). The mice were given the test preparations orally rather than intragastrically. Two tests (four mice per test) were conducted for each preparation, each test being carried out in a different consignment of mice. Negative controls consisted of broth culture supernatants and soft agar culture extract from a nonenterotoxigenic E. coli strain. 1W. E. Saunders Ltd., Downsview, Ontario.
Volume 40-July, 1976
RESULTS LIVE CULTURES
Piglet Gut Loops - Of the 200 isolates from scouring calves, 55 were positive in three or four and 70 were positive in one or two of the initial four tests. Two additional tests were conducted for each of the latter isolates and the results used to form Groups IIa and lIb (Table I). Among the isolates from normal calves, 85 were negative in all four tests (Table I) and 15 were positive in one or two of the six Lests (Table I). MRI were calculated for each group and the data analyzed by means of an analysis of variance test at the 95 percent confidence level. In view of the relatively high MRI values and the frequency of positive reactions, isolates of Groups I and IIa were considered enterotoxigenic. Those of Group Ilb were considered of doubtful enterotoxigenicity and those of Group III were considered nonenterotoxigenic. Calf Gut Loops - At post mortem, in most calves, almost all segments contained some fluid. However, positive segments were markedly distended and could be readily identified. Isolates were differentiated on the basis of the distention caused by them in comparison with the control segments as well as on MRI values. Segments that were markedly distended were regarded as positive, whereas segments which contained volumes of fluid similar to that in negative controls were considered negative. Isolates were found to give either positive reactions in all tests or negative reactions in all tests. All isolates found positive on visual comparison had MRI values ' 67 and those negative on visual comparison had MRI values < 28. Of the 200 from scouring calves, 55 had values > 67. One isolate from a normal calf had an MRI value . 67. There was a significant difference between isolates that had MRI values . 67 and those which had MRI values . 28 (t-test at 95 % confidence limits). Based on visual comparison and statistical analysis the 56 isolates with MRI values > 67 were classified as enterotoxigenic. The remaining 244 were categorized as nonenterotoxigenic. It was observed that fluid volumes associated with negative cultures varied with the area of intestine in which tests were conducted. Negative cultures in the upper
243
small intestine were associated with more fluid than those in the lower small intestine. However, the volume of fluid associated with negative cultures paralleled that of the negative control segment in that area. The results obtained from testing 300 isolates as live cultures in piglet and calf gut loops showed that 83 isolates gave some indication of enterotoxigenicity in one or both test systems. Forty-five of the 83 were enterotoxigenic in both test systems. Twenty-seven isolates classified enterotoxigenic in the piglet were found nonenterotoxigenic in the calf. Eleven isolates doubtful in the piglet were found enterotoxigenic in the calf. It was apparent that the results of piglet and calf gut loop systems did not correlate. Therefore, it was decided to test in vitro enterotoxin production by the 83 isolates that had given some indication of enterotoxigenicity. TEST OF ISOLATES FOR in vitro ENTEROTOXIN PRODUCTION
Piglet gut loops - Of the 83 preparations tested, 45 were positive in three or four of four tests conducted and had MRI values . 61. Twenty-two did not give positive reactions in the heated form but were positive twice in the unheated form. They had MRI values . 39. The remaining 16 were negative in all four tests. The 45 prepara-
tions with MRI values . 61 were found to cause significantly more fluid accumulation than the 22 with MRI values . 39. The former were therefore considered enterotoxigenic and the latter of doubtful enterotoxigenicity. The remaining 16 were classified nonenterotoxigenic. Calf Gut Loop - The 83 preparations were subjected to one test each in the unheated form. Fifty-six, including one isolate from a normal calf, were found visually positive and had MRI values . 60. The remaining 27 were visually negative and had MRI values . 33. Statistically, the former preparations were found to cause significantly more fluid accumulation than the latter (t-test 95 percent confidence limits). The 56 preparations with MRI values . 60 were therefore classified enterotoxic and the remaining 27 were considered nonenterotoxic. Four test preparations which were enterotoxic in the unheated form were tested in the heated form. There was no significant difference between the MRI values of the heated and unheated forms of these preparations.
Suckling Moutse Test - The 83 BS preparations tested in the piglet and calf gut loop systems were used. BS preparations were graded for enterotoxic activity based on gut weight to body weight ratios. Preparations that gave ratios < 0.070 were
TABLE I. Classification of Bovine E. coli on the Basis of Gut-Loop Tests in Piglets
Group I (3/4 or 4/4)a ............................. IIa (3/6 or 4/6).............................. IIb (1/6 or 2/6)..............................
III (0/4). Total .................................
No. of Isolates from Scouring Calves 55 17 55 73 200
No. of Isolates from Normal Calves 0 0 15 85 100
aNumber positive Number of tests TABLE II. Distribution of Mean Gut Weight: Body Weight Ratios Obtained with 83 Broth Supernatant Preparations Tested in Suckling Mice Meana Gut Weight: Body Weight > 0.090 (x = 0.132)................................. 0.070 - 0.090 (x = 0.075)............................ < 0.070 (x = 0.059)..................................
No. of Preparations Interpretation 52 Enterotoxic 16 Questionably
15
enterotoxic Nonenterotoxic
aMean of two tests
bi is the mean for all tests falling in the category
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Can. J. comp. Med.
TABLE III. Comparison of the Enterotoxicity of 83 Bovine E. coli Isolates in the Piglet, Calf and Mouse Test Systems
Number Reacting in Piglet and Calf Test Systems 72 ..........................45 Piglet Calf 56 ..........................56 45 ...........................45 Piglet and calf 27a .0 Piglet only Calf only llb ...........................
Number Reacting in Mice Questionable Negative 15 12
Positive
11
0 0 15 0
0 0 12 0
aIsolates found enterotoxigenic in the piglet but not in the calf blsolates found doubtful in the piglet but enterotoxigenic in the calf considered negative, those in the range of 0.070 to 0.900 were questionably positive and those . 0.090 were positive (2). The ranges of ratios obtained are indicated in Table II. Questionably positive BS preparations were concentrated and retested. Four of the 16 concentrated preparations were positive. SA and concentrated SA preparations were made from the remaining 12 whose concentrated BS preparations were questionably positive. In all tests these preparations remained questionably positive. In Table III, a comparison of results of the tests of enterotoxin preparations in all the three test systems is presented.
rhea. They believed that information, however, might be misleading when applied to animals of a different species from the ones in which the test had been performed. It appears, therefore, that the results obtained from testing bovine E. coli in calf gut loops would be more representative of the situation with regard to the existence of enterotoxigenic types of E. coli within the population of the 300 strains tested. In the calf intestine, there was a clearcut differentiation of the 300 isolates into enterotoxigenic and nonenterotoxigenic groups. The response was very consistent, an isolate being either always positive or always negative. It appears that certain strains of E. coli capable of surviving and producing enterotoxin in piglet gut loops are incapable of DISCUSSION enterotoxic activity in calf gut loops and under the conditions of in vitro growth The attempt to use the piglet gut loop used in the mouse test system. This is model for evaluating enterotoxigenicity of evident from the observation of the 72 bovine E. coli was unsuccessful. This model isolates found enterotoxigenic in the piglet failed to differentiate the isolates into an gut loop test system, where 27 were nonenterotoxigenic and nonenterotoxigenic enterotoxigenic in calf gut loops and were group. The results are in marked contrast either questionably positive or negative in to those obtained with E. coli of porcine the mouse test system. Isolates found nonorigin (6, 7, 13). Seventy of the 300 iso- enterotoxigenic in the piglet were also nonlates gave infrequent positive reactions in enterotoxigenic in the calf gut loop. Eleven isolates classified doubtful in the the piglet and were considered of doubtful enterotoxigenicity. Subsequent tests in the piglet were enterotoxigenic in the calf and calf indicated that only 11 of these were mouse test systems indicating that strains enterotoxigenic. These reactions may there- of E. coli may be poorly enterotoxigenic fore represent false positive reactions or in the piglet but highly enterotoxigenic responses to a product not significant in when tested in the calf and mouse test systems. the calf intestine. Tests of BS preparations indicated no Smith and Halls (13) studied E. coli from pigs, calves, lambs, rabbits and man differences in the degree of responsiveness by the ligated segment and oral inoculation of the piglet gut loop system to heated and methods. Their results indicated that the unheated forms of 45 BS preparations clasreaction of dilation produced by a strain- sified as possessing enterotoxin activity. It of E. coli in a ligated segment of small therefore appears that E. coli of bovine intestine provided useful information on origin produce an enterotoxin that rethe ability of the strain to produce diar- sembles the heat stable (ST) rather than
Volume 40 -July, 1976
245
the heat labile (LT) form of enterotoxin produced by typical pig enteropathogenic E. coli strains. This is consistent with the findings of Smith and Linggood (15). This is also supported by the fact that no difference was observed in the degree of responsiveness of the calf gut loop system to unheated and heated forms of four enterotoxigenic preparations. One hundred percent correlation was seen between tests of live cultures in calf gut loops and tests of broth supernatant in the mouse. The mouse test system is therefore a valuable indicator of enterotoxigenicity of bovine strains of E. coli. However, BS preparations from four isolates had to be concentrated before they were shown to give positive responses. These four strains tested as cultures in calf gut loops were observed to cause marked distension. This observation is consistent with the fact that certain E. coli strains are poor enterotoxin producers in vitro, although they may possess good enterotoxic activity in vivo (6). BS preparations with low enterotoxin content could therefore give rise to questionably positive reactions in the mouse test system. Concentration followed by retesting seems to be valuable in confirming the enterotoxic activity of test preparations found questionably positive in the mouse test system. It is therefore concluded from this study that for purposes of testing bovine E. coli for enterotoxigenicity, the calf and the mouse test systems are most suitable. The latter offers greater convenience when testing a small number of isolates. The calf gut loop test system has been found useful for determining the enterotoxigenicity of bovine E. coli by previous workers (11). Smith and Halls (14) suggested that only enterotoxigenic strains of E. coli can act as primary enteropathogens in the calf diarrhea syndrome. The data from the calf gut loop and mouse test systems used in this study indicate that only 27.5% of E. coli isolated from scouring calves were enterotoxigenic. These results are similar to those reported by Acres and co-workers (1) who found that 30 percent of the strains isolated from calves were diarrheic feces dilated calf gut loops. In that study (1) diarrhea was reproduced in young lambs with strains of E. coli that dilated calf gut, demonstrating as Smith and Halls (13) had done, that there is a good correlation between loop dilating activity and ability to cause diarrhea.
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ACKNOWLEDGMENTS The authors are grateful to Dr. D. A. Barnum and Dr. D. G. Butler for their advice and assistance throughout the study. Technical assistance was provided by Miss M. Wilke. The research was supported by a grant from the National Research Council and by the Ontario Ministry of Agriculture and Food.
REFERENCES 1. ACRES, S. D., C. J. LAING, J. R. SAUNDERS and 0. M. RADOSITIS. Acute undiffer entiated neonatal diarr hea in beef calves I. Occurrl ence and distribution of infectious agents. Can. J. comp. Med. 39: 116-132. 1975. 2. DEAN, A. G., Y. C. CHING, R. G. WILLIAMS and L. B. HARDEN. Test for Escherichia coli enterotoxin using infant mice: Application in a study of diar rhea in childr en in Honolulu. J. infect. Dis. 125: 407-411. 1972. 3. DUNNE, H. W. An expanding concept of colibacillosis with emphasis on the disease in swine. Can. J. comp. Med. 23: 101-104. 1959. 4. GLANTZ, P. J., H. W. DUNNE, C. E. HEIST and J. F. HOKANSON. Bacteriological and serological studies of Escherichia coli serotypes associated with calf scours. Penn. State Univ. Agr. Exp. Stat. Bull. 645: 1-22. 1959. 5. GOSSLING, J., K. A. McKAY and D. A. BARNUM. Colibacillosis of calves in Ontario II. The association of certain serotypes of Eacherichia coli with calf scours. Can. vet. J. 5: 220-228. 1964. 6. GYLES, C. L. The response of ligated loops of pigs intestine to Escherichia coli. Thesis. Univ. of Guelph. 1966. 7. GYLES, C. L. and D. A. BARNUM. Escherichia coli in ligated segments of pig intestine. J. Path. 29: 189-194. 1967. 8. KOHLER, E. M. Enterotoxic activity of filtrates of Escherichia coli in young pigs. Am. J. vet. Res. 29: 2263-2274. 1968. 9. MEBUS, C. A., N. R. UNDERDAHL, M. B. RHODES and M. J. TWIEHAUS. Calf diarrhea (Scours : Reproduced with a virus from a field outbreak. Univ. Nebraska. Res. Bull. No. 223: 1-16. 1969. 10. MEBUS, C. A., M. KOND, N. R. UNDERDAHL and M. J. TWIEHAUS. Cell culture propagation of neonatal calf diar rhea (scours) virus. Can. vet. J. 12: 69-72. 1971. 11. MYERS, L. L., F. S. NEWMAN, G. R. WARREN, J. E. CATLIN and C. K. ANDERSON. Calf ligated intestinal segment test to detect enterotoxigenic Escherichia coli. Infection & Immunity 11: 588-591. 1975. 12. SIVASWAMY, G. Studies on enterotoxigenic bovine Escherichia coli. Thesis. Univ. of Guelph. 1975. 13. SMITH, H. W. and S. HALLS. Observations by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Path. 93: 499-529. 1967. 14. SMITH, H. W. and S. HALLS. Studies on Escherichia coli enterotoxin. J. Path. 93: 531-543. 1967. 15. SMITH, H. W. and M. A. LINGGOOD. Further observations on Escherichia coli enterotoxins with particular regards to those produced by atypical piglet strains and by calf and lamb strains. The transmissible nature of these enterotoxins and of a K antigen possessed by calf and lamb strains. J. med. Microbiol. 5: 243-250. 1972. 16. STEVENS. J. B. Experimental diarrhea in pigs exposed to Eacherichia coli enterotoxin. Thesis. Univ. of Guelph. 1971. 17. WRAY, C. and J. R. THOMLINSON. Factors influencing occurrence of colibacillosis in calves. Vet. Rec. 96: 52-56. 1975. 18. WHITE, R. G., C. A. MEBUS and M. J. TWIEHAUS. Incidence of herds infected with neonatal calf diarrhea virus (NCDV). Vet. Med. small Anim. Clin: 65: 487-490. 1970.
Can. J. comp. Med.
que celui de 100 autres souches d'E. coli isolees d'un groupe de veaux temoins et normaux. Les auteurs utiliserent les trois m6thodes suivantes pour verifier l'enterotoxigenicite' des cultures: l'inoculation de segments d'intestin ligatures, chez des porcelets et des veaux, ainsi que l'injection orale 'a des souriceaux. Ils utiliserent des cultures vivantes de tous les colibacilles pour l'inoculation des segments d'intestin ligatures, tandis qu'ils employierent le surnageant sterile du bouillon des cultures pour l'injection or-ale 'a des souriceaux. Des souches isolees des veaux atteints de diarrhee, 36% s'avererent enterotoxinogenes dans l'intestin du porcelet et 28%, dans celui du veau. Aucune des souches isolees des veaux temoins ne s 'avera enterotoxinogiene dans l'intestin du porcelet; l'une d'elles s'avera cependant enterotoxinogene dans celui du veau. La methode des ligatures de l'intestin du porcelet sembla impropre at la verification du pouvoir enterotoxinogiene des souches bovines d'E. coli; par ailleurs, celle des ligatures de l'intestin du veau s'avera satisfaisante et corrobora parfaitement les resultats obtenus avec les souriceaux. Les E. coli enterotoxinogenes d'origine bovine produisirent une enterotoxine semblable a l'enterotoxine thermostable des souches d'E. coli enteropathogenes porcines typiques.
INTRODUCTION
RESUME Cette experience visait 'a determiner le pouvoir enterotoxinogZene de 200 souches d'Escherichia coli isolees de veaux ages de moins de deux semaines et atteints de diarrhee, ainsi
*Department of Veterinary Microbiology and Immunology, Ontario Veterinary College Univeraity of Guelph, Guelph, Ontario NIG 2W1. Submitted September 24, 1975.
Volume 40 -July, 1976
A wide variety of infectious and noninfectious agents have been implicated as causes of diarrhea in young calves (1, 3, 4, 9, 13, 17). Among the infectious agents, specific viruses have recently been recognized as etiological agents of calf diarrhea (1, 9, 10, 18) and the bacterium E. coli has for a long time been considered an important cause of calf scours (4, 13). The recognition that E. coti which cause diarrhea in calves are characterized by their production of enterotoxin (14) has paved the way for studies to evaluate the importance of E. coli among the various
241
agents implicated in calf diarrhea. Determination of the prevalence of enterotoxigenic E. coli in the feces of calves with diarrhea is an effective way of assessing the importance of E. coli in the calf diarrhea syndrome. In one study conducted in Great Britain, Smith and Halls (13) suggested that only a small percentage of cases of calf scours is attributable to E. coli. The objective of this study, therefore, was to determine the frequency of occurrence of enterotoxicity among E. coli isolates from the feces of scouring and normal calves. For this purpose, three test systems were used. Initially, the piglet gut loop test system was used since the ligated intestine of young piglets appeared to respond to enterotoxigenic E. coli of bovine origin (15). Subsequently, the cultures were tested in ligated intestine of calves. Finally, tests for in vitro enterotoxin production were conducted in piglet and calf gut loops and in suckling mice.
BROTH CULTURES
Broth cultures for gut loop tests were prepared by inoculating organisms into Tryptic Soy Broth (TSB) in 20 ml screw capped bottles, which were incubated at 37°C for 18 hours in a water bath at a rotary speed of 150 r.p.m. ENTEROTOXIN PREPARATION
Soft agar (SA) and broth supernatant (BS) preparations were as described by Stevens (16). CONCENTRATION OF ENTEROTOXIN PREPARATION
Concentration involved precipitation followed by extraction. The procedure for precipitation was similar to that described by Smith and Halls (14). GUT LooP TEST
MATERIALS AND METHODS
Piglets - The surgical procedure followed was similar to that described by Gyles and Barnum (7). For testing live ANIMALS cultures, 20 to 30 segments were made in each pig and the first segment was infed and colostrum hundred sixty One jected with normal saline. The segments in seven day old piglets and 13 five day old colostrum fed calves were used for ligated each pig were organized into seven segintestine tests. Four day old suckling mice ment test areas with a central positive (Connaught Medical Research Laboratory control. The volume of inoculum was 1.5 strain) were used in tests for enterotoxi- ml. At post mortem the results were read by qualitative and quantitative methods. city. Segments with fluid were positive and those without fluid were negative. For quantitative evaluation, the fluid volumes CULTURES per cm of intestine for positive segments Three hundred strains of E. coli, 200 in a test area were expressed as percentfrom scouring calves and 100 from normal ages compared with the value of the positive control. The percentage was called the ones, were isolated from the feces of calves under two weeks of age. The strains were reaction index (RI) and a mean reaction isolated over the period 1969 to 1974 and index (MRI) was calculated for each isostored on Trypticase Soy Agar (TSA) late. Each isolate was initially tested four slants. The isolates were subjected to bio- times. Two additional tests were conducted chemical tests to confirm their identity as for isolates found positive in one or two E. coli. Escherichia coli strains PS274 (4) of the initial four tests. For testing BS preparations, eight to and Y97 which had previously been shown segments were made in each piglet. twelve of calves (Gyles, 1973, to dilate gut loops unpublished), were used as positive con- Each BS preparation was tested a total trol organisms when testing live cultures of four times, twice in the unheated form of E. coli. Enterotoxin preparation from and twice after treatment at 65°C for 15 strain Y97 was used as a positive control minutes. The volume of inoculum was 8 ml. Heated and unheated forms of BS preparawhen testing enterotoxin preparations.
242
Can. J. comp. Med.
tions were tested in adjacent segments in the same pig. Calves - Each calf was starved for 12 to 24 hours prior to surgery. Anaesthesia was induced and maintained with halothane and a laparotomy was carried out aseptically through a paramedian incision on the right side. Silk ligatures which occluded the lumen were placed throughout the small intestine, beginning approximately one metre from the pylorus. Sixty to ninety segments were made in each calf and 2 ml broth culture was injected into each segment. Throughout surgery the calf was given intravenously between one and two litres of a balanced electrolyte solution (Proionate'). Segments of intestine were organized in series of 11 segment test areas with a central positive control. A negative control segment injected with TSB was included following every 22 segments. Three tests (each in a different calf) were conducted for each of the isolates classified enterotoxigenic in the piglet system. Isolates nonenterotoxigenic or doubtful in the piglet system were subjected initially to two tests but were subjected to an additional test when positive reactions were obtained. BS preparations were tested in the anterior 140-150 cm of the area of intestine selected for ligation. In each calf, 14 or 15 segments were prepared and injected with 8 ml of BS preparation. The seventh segment was injected with the negative control and the last segment of the series with the positive control. Each BS preparation was tested once in the unheated form and four preparations were also evaluated in paired tests of heated (65°C for 15 minutes) and unheated BS preparations. SUCKLING MOUSE TEST The procedure followed was similar to that described by Dean and co-workers (2). The mice were given the test preparations orally rather than intragastrically. Two tests (four mice per test) were conducted for each preparation, each test being carried out in a different consignment of mice. Negative controls consisted of broth culture supernatants and soft agar culture extract from a nonenterotoxigenic E. coli strain. 1W. E. Saunders Ltd., Downsview, Ontario.
Volume 40-July, 1976
RESULTS LIVE CULTURES
Piglet Gut Loops - Of the 200 isolates from scouring calves, 55 were positive in three or four and 70 were positive in one or two of the initial four tests. Two additional tests were conducted for each of the latter isolates and the results used to form Groups IIa and lIb (Table I). Among the isolates from normal calves, 85 were negative in all four tests (Table I) and 15 were positive in one or two of the six Lests (Table I). MRI were calculated for each group and the data analyzed by means of an analysis of variance test at the 95 percent confidence level. In view of the relatively high MRI values and the frequency of positive reactions, isolates of Groups I and IIa were considered enterotoxigenic. Those of Group Ilb were considered of doubtful enterotoxigenicity and those of Group III were considered nonenterotoxigenic. Calf Gut Loops - At post mortem, in most calves, almost all segments contained some fluid. However, positive segments were markedly distended and could be readily identified. Isolates were differentiated on the basis of the distention caused by them in comparison with the control segments as well as on MRI values. Segments that were markedly distended were regarded as positive, whereas segments which contained volumes of fluid similar to that in negative controls were considered negative. Isolates were found to give either positive reactions in all tests or negative reactions in all tests. All isolates found positive on visual comparison had MRI values ' 67 and those negative on visual comparison had MRI values < 28. Of the 200 from scouring calves, 55 had values > 67. One isolate from a normal calf had an MRI value . 67. There was a significant difference between isolates that had MRI values . 67 and those which had MRI values . 28 (t-test at 95 % confidence limits). Based on visual comparison and statistical analysis the 56 isolates with MRI values > 67 were classified as enterotoxigenic. The remaining 244 were categorized as nonenterotoxigenic. It was observed that fluid volumes associated with negative cultures varied with the area of intestine in which tests were conducted. Negative cultures in the upper
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small intestine were associated with more fluid than those in the lower small intestine. However, the volume of fluid associated with negative cultures paralleled that of the negative control segment in that area. The results obtained from testing 300 isolates as live cultures in piglet and calf gut loops showed that 83 isolates gave some indication of enterotoxigenicity in one or both test systems. Forty-five of the 83 were enterotoxigenic in both test systems. Twenty-seven isolates classified enterotoxigenic in the piglet were found nonenterotoxigenic in the calf. Eleven isolates doubtful in the piglet were found enterotoxigenic in the calf. It was apparent that the results of piglet and calf gut loop systems did not correlate. Therefore, it was decided to test in vitro enterotoxin production by the 83 isolates that had given some indication of enterotoxigenicity. TEST OF ISOLATES FOR in vitro ENTEROTOXIN PRODUCTION
Piglet gut loops - Of the 83 preparations tested, 45 were positive in three or four of four tests conducted and had MRI values . 61. Twenty-two did not give positive reactions in the heated form but were positive twice in the unheated form. They had MRI values . 39. The remaining 16 were negative in all four tests. The 45 prepara-
tions with MRI values . 61 were found to cause significantly more fluid accumulation than the 22 with MRI values . 39. The former were therefore considered enterotoxigenic and the latter of doubtful enterotoxigenicity. The remaining 16 were classified nonenterotoxigenic. Calf Gut Loop - The 83 preparations were subjected to one test each in the unheated form. Fifty-six, including one isolate from a normal calf, were found visually positive and had MRI values . 60. The remaining 27 were visually negative and had MRI values . 33. Statistically, the former preparations were found to cause significantly more fluid accumulation than the latter (t-test 95 percent confidence limits). The 56 preparations with MRI values . 60 were therefore classified enterotoxic and the remaining 27 were considered nonenterotoxic. Four test preparations which were enterotoxic in the unheated form were tested in the heated form. There was no significant difference between the MRI values of the heated and unheated forms of these preparations.
Suckling Moutse Test - The 83 BS preparations tested in the piglet and calf gut loop systems were used. BS preparations were graded for enterotoxic activity based on gut weight to body weight ratios. Preparations that gave ratios < 0.070 were
TABLE I. Classification of Bovine E. coli on the Basis of Gut-Loop Tests in Piglets
Group I (3/4 or 4/4)a ............................. IIa (3/6 or 4/6).............................. IIb (1/6 or 2/6)..............................
III (0/4). Total .................................
No. of Isolates from Scouring Calves 55 17 55 73 200
No. of Isolates from Normal Calves 0 0 15 85 100
aNumber positive Number of tests TABLE II. Distribution of Mean Gut Weight: Body Weight Ratios Obtained with 83 Broth Supernatant Preparations Tested in Suckling Mice Meana Gut Weight: Body Weight > 0.090 (x = 0.132)................................. 0.070 - 0.090 (x = 0.075)............................ < 0.070 (x = 0.059)..................................
No. of Preparations Interpretation 52 Enterotoxic 16 Questionably
15
enterotoxic Nonenterotoxic
aMean of two tests
bi is the mean for all tests falling in the category
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TABLE III. Comparison of the Enterotoxicity of 83 Bovine E. coli Isolates in the Piglet, Calf and Mouse Test Systems
Number Reacting in Piglet and Calf Test Systems 72 ..........................45 Piglet Calf 56 ..........................56 45 ...........................45 Piglet and calf 27a .0 Piglet only Calf only llb ...........................
Number Reacting in Mice Questionable Negative 15 12
Positive
11
0 0 15 0
0 0 12 0
aIsolates found enterotoxigenic in the piglet but not in the calf blsolates found doubtful in the piglet but enterotoxigenic in the calf considered negative, those in the range of 0.070 to 0.900 were questionably positive and those . 0.090 were positive (2). The ranges of ratios obtained are indicated in Table II. Questionably positive BS preparations were concentrated and retested. Four of the 16 concentrated preparations were positive. SA and concentrated SA preparations were made from the remaining 12 whose concentrated BS preparations were questionably positive. In all tests these preparations remained questionably positive. In Table III, a comparison of results of the tests of enterotoxin preparations in all the three test systems is presented.
rhea. They believed that information, however, might be misleading when applied to animals of a different species from the ones in which the test had been performed. It appears, therefore, that the results obtained from testing bovine E. coli in calf gut loops would be more representative of the situation with regard to the existence of enterotoxigenic types of E. coli within the population of the 300 strains tested. In the calf intestine, there was a clearcut differentiation of the 300 isolates into enterotoxigenic and nonenterotoxigenic groups. The response was very consistent, an isolate being either always positive or always negative. It appears that certain strains of E. coli capable of surviving and producing enterotoxin in piglet gut loops are incapable of DISCUSSION enterotoxic activity in calf gut loops and under the conditions of in vitro growth The attempt to use the piglet gut loop used in the mouse test system. This is model for evaluating enterotoxigenicity of evident from the observation of the 72 bovine E. coli was unsuccessful. This model isolates found enterotoxigenic in the piglet failed to differentiate the isolates into an gut loop test system, where 27 were nonenterotoxigenic and nonenterotoxigenic enterotoxigenic in calf gut loops and were group. The results are in marked contrast either questionably positive or negative in to those obtained with E. coli of porcine the mouse test system. Isolates found nonorigin (6, 7, 13). Seventy of the 300 iso- enterotoxigenic in the piglet were also nonlates gave infrequent positive reactions in enterotoxigenic in the calf gut loop. Eleven isolates classified doubtful in the the piglet and were considered of doubtful enterotoxigenicity. Subsequent tests in the piglet were enterotoxigenic in the calf and calf indicated that only 11 of these were mouse test systems indicating that strains enterotoxigenic. These reactions may there- of E. coli may be poorly enterotoxigenic fore represent false positive reactions or in the piglet but highly enterotoxigenic responses to a product not significant in when tested in the calf and mouse test systems. the calf intestine. Tests of BS preparations indicated no Smith and Halls (13) studied E. coli from pigs, calves, lambs, rabbits and man differences in the degree of responsiveness by the ligated segment and oral inoculation of the piglet gut loop system to heated and methods. Their results indicated that the unheated forms of 45 BS preparations clasreaction of dilation produced by a strain- sified as possessing enterotoxin activity. It of E. coli in a ligated segment of small therefore appears that E. coli of bovine intestine provided useful information on origin produce an enterotoxin that rethe ability of the strain to produce diar- sembles the heat stable (ST) rather than
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the heat labile (LT) form of enterotoxin produced by typical pig enteropathogenic E. coli strains. This is consistent with the findings of Smith and Linggood (15). This is also supported by the fact that no difference was observed in the degree of responsiveness of the calf gut loop system to unheated and heated forms of four enterotoxigenic preparations. One hundred percent correlation was seen between tests of live cultures in calf gut loops and tests of broth supernatant in the mouse. The mouse test system is therefore a valuable indicator of enterotoxigenicity of bovine strains of E. coli. However, BS preparations from four isolates had to be concentrated before they were shown to give positive responses. These four strains tested as cultures in calf gut loops were observed to cause marked distension. This observation is consistent with the fact that certain E. coli strains are poor enterotoxin producers in vitro, although they may possess good enterotoxic activity in vivo (6). BS preparations with low enterotoxin content could therefore give rise to questionably positive reactions in the mouse test system. Concentration followed by retesting seems to be valuable in confirming the enterotoxic activity of test preparations found questionably positive in the mouse test system. It is therefore concluded from this study that for purposes of testing bovine E. coli for enterotoxigenicity, the calf and the mouse test systems are most suitable. The latter offers greater convenience when testing a small number of isolates. The calf gut loop test system has been found useful for determining the enterotoxigenicity of bovine E. coli by previous workers (11). Smith and Halls (14) suggested that only enterotoxigenic strains of E. coli can act as primary enteropathogens in the calf diarrhea syndrome. The data from the calf gut loop and mouse test systems used in this study indicate that only 27.5% of E. coli isolated from scouring calves were enterotoxigenic. These results are similar to those reported by Acres and co-workers (1) who found that 30 percent of the strains isolated from calves were diarrheic feces dilated calf gut loops. In that study (1) diarrhea was reproduced in young lambs with strains of E. coli that dilated calf gut, demonstrating as Smith and Halls (13) had done, that there is a good correlation between loop dilating activity and ability to cause diarrhea.
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ACKNOWLEDGMENTS The authors are grateful to Dr. D. A. Barnum and Dr. D. G. Butler for their advice and assistance throughout the study. Technical assistance was provided by Miss M. Wilke. The research was supported by a grant from the National Research Council and by the Ontario Ministry of Agriculture and Food.
REFERENCES 1. ACRES, S. D., C. J. LAING, J. R. SAUNDERS and 0. M. RADOSITIS. Acute undiffer entiated neonatal diarr hea in beef calves I. Occurrl ence and distribution of infectious agents. Can. J. comp. Med. 39: 116-132. 1975. 2. DEAN, A. G., Y. C. CHING, R. G. WILLIAMS and L. B. HARDEN. Test for Escherichia coli enterotoxin using infant mice: Application in a study of diar rhea in childr en in Honolulu. J. infect. Dis. 125: 407-411. 1972. 3. DUNNE, H. W. An expanding concept of colibacillosis with emphasis on the disease in swine. Can. J. comp. Med. 23: 101-104. 1959. 4. GLANTZ, P. J., H. W. DUNNE, C. E. HEIST and J. F. HOKANSON. Bacteriological and serological studies of Escherichia coli serotypes associated with calf scours. Penn. State Univ. Agr. Exp. Stat. Bull. 645: 1-22. 1959. 5. GOSSLING, J., K. A. McKAY and D. A. BARNUM. Colibacillosis of calves in Ontario II. The association of certain serotypes of Eacherichia coli with calf scours. Can. vet. J. 5: 220-228. 1964. 6. GYLES, C. L. The response of ligated loops of pigs intestine to Escherichia coli. Thesis. Univ. of Guelph. 1966. 7. GYLES, C. L. and D. A. BARNUM. Escherichia coli in ligated segments of pig intestine. J. Path. 29: 189-194. 1967. 8. KOHLER, E. M. Enterotoxic activity of filtrates of Escherichia coli in young pigs. Am. J. vet. Res. 29: 2263-2274. 1968. 9. MEBUS, C. A., N. R. UNDERDAHL, M. B. RHODES and M. J. TWIEHAUS. Calf diarrhea (Scours : Reproduced with a virus from a field outbreak. Univ. Nebraska. Res. Bull. No. 223: 1-16. 1969. 10. MEBUS, C. A., M. KOND, N. R. UNDERDAHL and M. J. TWIEHAUS. Cell culture propagation of neonatal calf diar rhea (scours) virus. Can. vet. J. 12: 69-72. 1971. 11. MYERS, L. L., F. S. NEWMAN, G. R. WARREN, J. E. CATLIN and C. K. ANDERSON. Calf ligated intestinal segment test to detect enterotoxigenic Escherichia coli. Infection & Immunity 11: 588-591. 1975. 12. SIVASWAMY, G. Studies on enterotoxigenic bovine Escherichia coli. Thesis. Univ. of Guelph. 1975. 13. SMITH, H. W. and S. HALLS. Observations by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Path. 93: 499-529. 1967. 14. SMITH, H. W. and S. HALLS. Studies on Escherichia coli enterotoxin. J. Path. 93: 531-543. 1967. 15. SMITH, H. W. and M. A. LINGGOOD. Further observations on Escherichia coli enterotoxins with particular regards to those produced by atypical piglet strains and by calf and lamb strains. The transmissible nature of these enterotoxins and of a K antigen possessed by calf and lamb strains. J. med. Microbiol. 5: 243-250. 1972. 16. STEVENS. J. B. Experimental diarrhea in pigs exposed to Eacherichia coli enterotoxin. Thesis. Univ. of Guelph. 1971. 17. WRAY, C. and J. R. THOMLINSON. Factors influencing occurrence of colibacillosis in calves. Vet. Rec. 96: 52-56. 1975. 18. WHITE, R. G., C. A. MEBUS and M. J. TWIEHAUS. Incidence of herds infected with neonatal calf diarrhea virus (NCDV). Vet. Med. small Anim. Clin: 65: 487-490. 1970.
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