Necrotic Enteritis in Broil er Chickens 111. Reproduction of the Disease J. R. Long and R. B. Truscott*
ABSTRACT Lesions typical of necrotic enteritis could be produced experimentally in from 11-26% of broiler chickens consuming feed containing approximately 107 Clostridium perfringens per gram. Highest mortality was produced using isolates from field cases of necrotic enteritis which were reisolated from experimental cases in the laboratory. Penicillin in the drinking water at 100,000 I.U./litre completely prevented mortality whereas chloramphenicol at 110 mg/litre delayed the onset and reduced the number of deaths compared to controls.
RP:SUME Les auteurs ont reussi 'a provoquer des lesions typiques d'enterite necrotique chez 11 a 26% des poulets de gril auxquels ils servaient une moulee contenant environ 107 Clostridium perfringens/g. Ils obtinrent le taux de mortalite le plus &leve' lorsqu'ils utilisaient des souches isolees de cas naturels ayant declenche la maladie chez des poulets inocules experimentalement au laboratoire. L'addition de penicilline a l'eau de boisson, 'a raison de 100,000 U.L/litre, enraya completement la mortalite, tandis que l'addition de 110 mg de chloramphenicol/litre retarda l'eclosion de la maladie et diminua le nombre de mortalite, comparativement aux poulets temoins.
*Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada NIG 2W1. Present address of senior author: Nova Scotia Department of Agriculture, Truro, Nova Scotia B2N 5E3. This work was supported by the Canadian Hatchery Federation. Pfizer Agricultural Division, Montreal and the Ontario Ministry of Agriculture and Food. Submitted May 13, 1975.
Volume 40
-
January, 1976
INTRODUCTION
Parish (9) published the first report on necrotic enteritis (NE) in 1961. Since then the disease has been reported from several countries and the literature (6) and pathology (2, 5, 7) have recently been reviewed in detail. Although the etiology has not been well defined Clostridium perfringens is consistently associated with intestinal lesions (2, 7, 10). Published reports (1, 4, 8, 11) indicate only limited success when workers attempted to reproduce the disease experimentally. This paper reports the reproduction of NE in broiler chickens fed a commercial ration heavily inoculated with a broth culture of various strains of C. perfringens isolated from field cases.
MATERIALS AND METHODS ISOLATION OF C. perfringens A portion of intestine from a field case was opened as aseptically as possible and a loop used to scrape the necrotic area. This was streaked out on duplicate 5% bovine blood agar plates containing neomycin sulphate1 (200 ,ug/ml). The plates were incubated at 370C for 18-24 hours in a catalytic anaerobic jar using a Gas Pak2 hydrogen
'Calbiochem, La Jolla, California, U.S.A. 2BBL, Division of Becton, Dickinson and Co., Clarkson, Ontario.
53
generator. An isolated colony of C. perfringens (representative isolates were identified using established criteria, 12) was transferred to 300 ml of freshly prepared cooked meat medium3 (CMM) which was incubated at 37°C for 24 hours (stock culture). The purity of this culture was tested by plating on blood agar medium without antibiotics and incubating aerobically and anaerobically overnight at 37°C. This stock culture was held at room temperature and used as long as liquid could be obtained. It was subcultured to fresh CMM when the fluid was close to exhaustion. Five of the isolates were tested for toxin production using the method of Smith (12). The toxin type was determined using Wellcome clostridial diagnostic serum'. Except as indicated, thioglycollate broth5 was used for growing C. perfringens to be added to the feed. Sufficient medium was prepared for one week's use and it was steamed and cooled prior to inoculation. One-half ml of stock culture was used as inoculum per 100 ml of thioglycollate broth.
atures indicated. Unless otherwise stated, freshly infected feed was provided ad libitum daily for five days. Water was always available free choice.
NUMBER OF C. perfringens The numbers of C. penfringens per ml of thioglycollate broth prior to its addition to feed and the numbers of these organisms per gram of inoculated and incubated feed just before feeding were determined as follows. One ml of broth was added to 9 ml of phosphate buffered saline (PBS), then mixed and four 0.2 ml volumes of appropriate dilutions were placed on blood agar plates. One gram of feed was added to 99 ml of PBS, mixed, diluted and plated as above. All plates were incubated anaerobically overnight and the mean colony count per ml was determined. CHICKENS
FEED
Commercial chick starter6 was used throughout except for one experiment in which a laboratory prepared feed7 was used. This latter feed, in sealed preserving cans, was sterilized with 5 Mrads of gamma irradiation8. INOCULATION OF FEED
When broth cultures were added, weighed amounts of feed were prepared and broth was mixed in such a way as to provide an even distribution of the broth throughout the feed. The feed/broth ratio in this mixture was 1/1.25 (w/v) and when incubated this was done aerobically at the temper-
3Difco Laboratories, Detroit, Michigan, U.S.A. 4War-ner-Chilcott Laboratories, Toronto, Ontario. 5Thioglycollate Broth Brewer's modified, BBL, Division of Becton, Dickinson, Clarkson, Ontario. 6Master Feed Krums, Maple Leaf Mills Limited, Toronto, Ontario. IPrepared by Department of Animal and Poultry Science, University of Guelph. SAtomic Energy of Canada, Limited. Ottawa, Ontario.
54
Columbian rock chicks were provided by the Department of Animal and Poultry Science from a small flock maintained for experimental purposes. Chicks were started in a Petersime' battery brooder in an isolation room in the isolation facilities of the Department of Veterinary Microbiology and Immunology. They were transferred to heated brooders in another room in groups of ten to 25 birds for experimentation at two to four weeks of age. A group of control birds equal to at least 10% of the birds on test was maintained on normal feed and water either in the test room or in a separate room. Necropsies were performed on all dead birds. Gross lesions in the intestine were recorded and representative sections of affected areas were collected for histological examination and bacteriological culture.
RESULTS The isolates obtained and results of the tests to determine their toxin are shown in Table I. This table shows that type A toxin 9Petersime Incubator Company, Gettysbury, Ohio, U.S.A.
Can. J. comp. Med.
TABLE I. Toxin Production from Field Isolates of C. perfringens Isolate
2153
.............
74-379 . ......................... 74-590 . ......................... L45 .. 2934 .......................... 2945 ..... ......... -NT = No toxin detected ',ND Not done
Toxin NT-
NDb A A NT A
could be demonstrated from only three of the five strains tested. Initial experiments were carried out with groups of ten birds each. The cells recovered by centrifugation from 600 ml of a 15 hr thioglycollate culture of isolate 2153 were resuspended to 50 ml in phosphate buffered saline pH 7.0 and 1 ml amounts were inoculated into the crop by means of an 18 gauge teat cannula. Four doses were given at four hour intervals for one day. No deaths resulted and the intestines were normal when examined macroscopically three days postinoculation. The next experiment involved the use of a 24 hr thioglycollate broth culture of 2153 and feed containing 5 or 20% starch. Approximately 750 ml of broth culture was mixed with 600 g of feed and incubated aerobically for 24 hours. A like amount of feed was inoculated and incubated anaerobically. This feed was provided as the only feed to two week old chicks till consumed. The results as presented in Table II suggested that the addition of starch might be important in disease reproduction and a further experiment was done with varied levels of starch in the ration. Two hundred grams of feed was mixed with 250 ml of a 24 hr broth culture of 2153 and the mixture was incubated aerobically for 24 hrs. This was repeated for a total of four daily feedings. The results presented in Table III suggested that a level of 2% starch was the most satisfactory. Further tests were done with differing levels of starch with varied results, generally lesions and mortality were greatest with 2% starch and all further trials, except as indicated, were done with 2% starch in the feed. To test the effect of incubation time and temperature of broth and broth feed mixtures, broths were incubated for nine, 15 or 24 hrs at 33, 37 or 41°C and the broth feed mixtures were incubated for nine or
Volume 40
January, 1976
Fig. 1. Broiler intestine from a case of experimentally reproduced necrotic enteritis with a typical diphtheritic membrane.
15 hrs at these same temperatures. The birds were fed daily for five days. The results as presented in Table IV suggested that the most favourable results were obtained when the broth was incubated for 15 hrs at 37°C and the broth feed mixture was incubated for nine hrs at 33°C and this was adopted as a standard incubation time and temperature except as indicated. In an attempt to determine whether greater mortality might result if the test strains were freshly isolated and used for the next experiment, several tests were carried out. These were done to compare initial isolates from different field cases using the standard incubation conditions developed and further to test reisolates as designated by RI, etc. In addition, feed sterilized by irradiation was inoculated and incubated to determine whether similar results might be obtained. Also, to determine whether the medium
55
TABLE II. Comparison of Aerobic and Anaerobic Incubation of Broth Feed Mixture for Reproduction of Necrotic Enteritis % Starch Dead/Treated
Incubation
Aerobic
5
............
Aerobic .20 Anaerobic .20 -NE = Necrotic enteritis TABLE III. Effect of Varied Levels of Starch Mixed with Feed on the Reproduction of Necrotic Enteritis
% Starch
Dead/Treated
0.............. 1............ 2............ 5............ -NE = Necrotic enteritis
1/10 0/10 4/10 0/10
Remarks NE-
NE
influenced the results obtained, a series of birds received feed inoculated with thioglycollate broth U.S.P.10 The results as presented in Table V suggest that virulence may be increased by bird passage and reisolation. No further attempts were made with isolate 74-379 because of the low mortality produced. Similar results had occurred with isolates 2934 and 2945. Thioglycollate broth U.S.P. appeared to be as satisfactory as Brewer's modified for growth of the test strain. To test the prophylactic effect of penicillin and chloramphenicol" upon necrotic enteritis, groups of birds received L45-R2 infected feed prepared as above and were provided with water containing 100,000 I.U. penicillin/litre or 110 mg chloramphenicol/litre. Treated water was provided eight hours prior to infection exposure and continued for six days following the last feeding of infected feed. Fresh antibiotic containing water was provided every 12 hours throughout the experimental period. The results as shown in Table VI indicate that penicillin at the test level was effective in preventing mortality while chloramphenicol reduced and delayed mortality but did not prevent the disease. A further experiment was carried out to assess the effect of feed with and without starch using the standard incubation times
0OBBL, Thioglycollage broth U.S.P.
1Francomycin 125, P.V.U. Inc., Hamilton, Ontario.
56
2/10
3/10 2/10
Remarks
Lesions of NE-
Lesion suggestive of NE Intestine grossly normal
and also to compare the standard incubation of the broth feed mixture with unincubated inoculated feed and further to compare feeding for one, two or the usual five days. Isolate L45-R3 was used for this test and the broth was incubated for 15 hrs at 370C. The results as shown in Table VII indicate that a longer exposure to infection results in higher mortality. It does not appear that starch is necessary and furthermore incubation of the broth feed mixture did not produce as high mortality as did the inoculated feed that was not incubated.
Fig. 2. An early lesion of necrotic enteritis with necrosis in the lamina propria. Apex of villus at right. H and E. X400.
Can. J. comp. Med.
TABLE IV. Effect of Varied Incubation Time and Temperature on the Production of Necrotic Enteritis Using Strain 2153 Broth Incubation Time/hr Temp/C°
Feed Incubation Temp/C° Time/hr
33 9 33 33 15 33 33 37 15 33 24 33 37 9 37 37 37 15 37 33 15 24 37 37 41 9 41 24 41 41 -All dead birds had lesions typical of necrotic enteritis
9 9 9 15 15 9 9 15 15 15
Results
-Dead/Treated 0/20 3/20 2/20 3/20 4/40 1/20 6/20 2/40 1/20
4/20
TABLE V. Pathogenicity Tests of Various Isolates and the Effect of Reisolation
Isolates Dead/Treated % Mortality 6.9 2153R3....... 11/16Cc 13.7 2153R4 22/160 13.1 2153R5....... 21/160 1.0 74-379 1/100 9.0 74-590 9/100 17.0 74-590R1 17/100 19.0 L45 ......... 19/100 9.0 L458 ......... 9/100 26.0 L45R1....... 26/100 28.0 L45RIb 17/60 WFeed sterilized by Gamma irradiation bGrown in thioglycollate broth, U.S.P.. BBL All dead birds had lesions of NE .......
........
.......
of C. perfringens. Histological sections from intestinal lesions of birds infected with various isolates showed early (Fig. 2) and advanced lesions (Fig. 3) of NE. Representative colony counts are presented in Table VIII where it may be seen that although there were variations in the incubation times the differences in counts from broth cultures of the four strains shown does not appear to be great. Similarly with the counts from the feed, on occasion almost identical counts were obtained with three of the four strains used, although the variation between samples was found from Fig. 3. Necrotic enteritis with necrosis of all villus somewhat greater than those structures. H and E. X40. broth. It is also apparent that the counts from inoculated feed were markedly less than those from broth and much less than With the exception of two birds (Table can be accounted for on the basis of diluII) which died in early experiments, macro- tion by feed. Although not evident from scopic lesions typical of necrotic enteritis the table there did not appear to be a rela(Fig. 1) were observed in the intestine of tionship between the counts and the actiall birds which died following experimental vity obtained with respect to disease reinfection. Gram stain smears and cultures production for any particular inoculated from these lesions showed large numbers feed.
Volume 40
-
January, 1976
57
DISCUSSION
The intestinal lesions in the experimentally reproduced disease were identical macroscopically and microscopically to those observed in field cases (2, 5, 7). Occasionally deaths occurred within 24 hours after consuming inoculated feed but deaths were more common after 36 hours with the peak mortality being reached at 48 hours. Deaths continued to occur throughout the five day experimental period. In later experiments when birds were kept for five days beyond the usual five day
test period it was observed that a few deaths occurred for the next three days. The acute nature of the disease was often seen with sick birds dying within 30 minutes after illness was observed. The finding that exposure to C. perfringens for a 24 hour feeding period resulted in 12% mortality while a five day feeding period resulted in mortality as high as 26% suggests a relationship between continued exposure to the organisms and disease incidence (Table VII). However, since 26% mortality was the maximum achieved in these studies it is apparent that other factors, as yet undetermined, may be important in reproducing
TABLE VI. Effect of Penicillin and Chloramphenicol in the Prevention of Mortality Due to Necrotic Enteritis
Treated Controls %0 Antibiotic Trial Dead/Treated Dead/Treated % Mortality 1 Penicitlin ............. 0 17 0/100 17/100 Penicillin ............. 2 0 21 0/90 19/90 Chloramphenical ....... 1 12a 12/100 25 25/100 *First deaths occurred 48 hours after deaths had occurred in control birds TABLE VII. Comparison of Incubation Time. Level of Starch and Exposure Periods for the Production of Necrotic Enteritis
Incubation Temp(C°)/Time(hrs) 33 9 33 9 33 9 33 9 33 9 0 0
%0 Starch 2 2 2
0 0 0
No. Feedings (days) 1 2 5 5 5 5
% Mortality 12 11 17 14 19 26
Dead/Treated 11/92
10/92
17/100 14/100 19/100 26/100
TABLE VIII. Colony Counts of Four Cultures of Clostridium perfringens from Broth and Inoculated Feed
Broth
Isolates 2934
2153 L45 590
Hrs. Incub.9
Count/ml X
107 44
8
18
15 15 15 15 15
18 27 15 28 20
Feed
Hrs. Incub. 5 5 5 5 15 15 9 9 9 9 9
Tenirp.
Incub. 37 37 37 37 37 37 33 33 33 33 33
c7^
Starch 0 1 2 3 2 4
2
2 0 2 2
Count/g X
107 7.3 5.8 2.9 4.5 2.6 1.9 2.7 5.0 96 8.5
4.6
Incubated at 370C
58
Can. J. comp. Med.
the disease. In preliminary studies starch appeared to be useful in assisting in disease reproduction and it was used throughout most of these studies. In a few final experiments (Table VII) where it was omitted the results suggest that it was not necessary. The role of such additives can only be determined with a series of experiments comparing diets with and without such substances. This disease appears to be one in which if the disease is manifest the bird dies, since an examination of survivors indicated that the intestines of such birds were grossly normal, while organisms resembl-
ing C. perfringens could be readily found throughout the intestinal tract. It is postu-
lated that multiplication of C. perfringe'ns
to threshold numbers within a particular time period is one of the major requirements for disease to occur. Whether continued feeding of infected feed beyond the tested five day period would assist or whether the critical time is actually within this period remains to be determined. At any rate, the percent mortality in these studies does exceed those reported from the field (5, 6) and it is likely that the level and duration of exposure are important here. It is possible that with a prolonged low level exposure sufficient immunity could develop to cause the disease to be selflimiting within a flock. Although the pathogenesis of intestinal lesions has not been worked out a possible pathogenesis has been proposed (7). From these studies it appears that oral feeding
with a virulent strain of C. perft ingens will
allow the disease to be consistently reproduced. With this method of reproduction the exact mechanism of lesion production can be studied. From this (Table I) and previous studies (7) it seems that all iso-
lates from necrotic enteritis are C. perfrin-
gens type A or do not yield enough toxin in vitro to permit typing. Such isolates are considered to be type A (12). Penicillin added to the drinking water (Table VI) protected birds completely and
Volume 40 - January, 1976
this antibiotic is commonly used in Ontario to treat field cases. Chloramphenicol, at the level used, delayed and reduced but did not prevent mortality (Table VI). These findings may be explained on the basis that the action of chloramphenicol may have been incomplete in the intestine. However, all strains studied were susceptible in vitro to both antibiotics using the disc method.
ACKNOWLEDGMENTS The authors thank Dr. J. R. Pettit for his assistance with the microscopic examination of intestinal lesions and Dr. N. C. Palmer for help with photography. REFERENCES 1. BERNIER, G. and R. FILION. Necrotic enteiritis in br oiler chickens. J. Am. vet. med. Ass. 158: 18961897. 1971. 2. BERNIER, G., J. B. PHANEUF et R. FILION. Ent& rite necrotique chez le poulet de gril I. Aspects clinico-pathologique. Can. J. comp. Med. 38: 280285. 1974. 3. BERNIER, G., R. FILION, R. MALO et J. B. PHANEUF. Enterite necrotique chez le poulet de gril II. Caracteres des souches de Clostridium perfringens isolees. Can. J. comp. Med. 38: 286-291. 1974. 4. DAVIS, R. B., J. BROWN and D. L. DAWE. Quail -biological indicators in the differentiation of ulcerative and necrotic enteritis of chickens. Poult. Sci. 50: 737-740. 1971. 5. HELMBOLDT, C. F. and E. S. BRYANT. The pathology of necrotic enteritis in domestic fowl. Avian Dis. 15: 775-780. 1971. 6. LONG, J. R. Necrotic enteritis in broiler chickens I. A review of the literature and the prevalence of tne disease in Ontario. Can. J. comp. Med. 37: 302308. 1973. 7. LONG, J. R., J. R. PETTIT and D. A. BARNUM. Necrotic enteritis in broiler chickens II. Pathology and proposed pathogenesis. Can. J. comp. Med. 38: 467-474. 1974. 8. NAIRN, M. E. and V. W. BAMFORD. Necrotic enteritis of broiler chickens in western Australia. Aust. vet. J. 43: 49-54. 1967. 1. PARISH, W. E. Necrotic enteritis in the fowl. 1. Histopathology of the disease and isolation of a strain of Clostridium welchii. J. comp. Path. 71: 377-393. 1961. 10. PARISH, W. E. Necrotic enteritis in the fowl. II. Examination of the causal Clostridium welchii. J. comp. Path. 71: 394-404. 1961. 11. PARISH, W. E. Necrotic enteritis in the fowl. IHI. The experimental disease. J. comp. Path. 71: 405413. 1961. 12. SMITH. L. DS. Clostridia. In Manual of Clinical Microbiology. Eldited by J. E. Blair, E. H. Lennette and J. P. Truant. pp. 280-283. Bethesda, Maryland: American Society of Microbiology. 1970.
59
ABSTRACT Lesions typical of necrotic enteritis could be produced experimentally in from 11-26% of broiler chickens consuming feed containing approximately 107 Clostridium perfringens per gram. Highest mortality was produced using isolates from field cases of necrotic enteritis which were reisolated from experimental cases in the laboratory. Penicillin in the drinking water at 100,000 I.U./litre completely prevented mortality whereas chloramphenicol at 110 mg/litre delayed the onset and reduced the number of deaths compared to controls.
RP:SUME Les auteurs ont reussi 'a provoquer des lesions typiques d'enterite necrotique chez 11 a 26% des poulets de gril auxquels ils servaient une moulee contenant environ 107 Clostridium perfringens/g. Ils obtinrent le taux de mortalite le plus &leve' lorsqu'ils utilisaient des souches isolees de cas naturels ayant declenche la maladie chez des poulets inocules experimentalement au laboratoire. L'addition de penicilline a l'eau de boisson, 'a raison de 100,000 U.L/litre, enraya completement la mortalite, tandis que l'addition de 110 mg de chloramphenicol/litre retarda l'eclosion de la maladie et diminua le nombre de mortalite, comparativement aux poulets temoins.
*Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada NIG 2W1. Present address of senior author: Nova Scotia Department of Agriculture, Truro, Nova Scotia B2N 5E3. This work was supported by the Canadian Hatchery Federation. Pfizer Agricultural Division, Montreal and the Ontario Ministry of Agriculture and Food. Submitted May 13, 1975.
Volume 40
-
January, 1976
INTRODUCTION
Parish (9) published the first report on necrotic enteritis (NE) in 1961. Since then the disease has been reported from several countries and the literature (6) and pathology (2, 5, 7) have recently been reviewed in detail. Although the etiology has not been well defined Clostridium perfringens is consistently associated with intestinal lesions (2, 7, 10). Published reports (1, 4, 8, 11) indicate only limited success when workers attempted to reproduce the disease experimentally. This paper reports the reproduction of NE in broiler chickens fed a commercial ration heavily inoculated with a broth culture of various strains of C. perfringens isolated from field cases.
MATERIALS AND METHODS ISOLATION OF C. perfringens A portion of intestine from a field case was opened as aseptically as possible and a loop used to scrape the necrotic area. This was streaked out on duplicate 5% bovine blood agar plates containing neomycin sulphate1 (200 ,ug/ml). The plates were incubated at 370C for 18-24 hours in a catalytic anaerobic jar using a Gas Pak2 hydrogen
'Calbiochem, La Jolla, California, U.S.A. 2BBL, Division of Becton, Dickinson and Co., Clarkson, Ontario.
53
generator. An isolated colony of C. perfringens (representative isolates were identified using established criteria, 12) was transferred to 300 ml of freshly prepared cooked meat medium3 (CMM) which was incubated at 37°C for 24 hours (stock culture). The purity of this culture was tested by plating on blood agar medium without antibiotics and incubating aerobically and anaerobically overnight at 37°C. This stock culture was held at room temperature and used as long as liquid could be obtained. It was subcultured to fresh CMM when the fluid was close to exhaustion. Five of the isolates were tested for toxin production using the method of Smith (12). The toxin type was determined using Wellcome clostridial diagnostic serum'. Except as indicated, thioglycollate broth5 was used for growing C. perfringens to be added to the feed. Sufficient medium was prepared for one week's use and it was steamed and cooled prior to inoculation. One-half ml of stock culture was used as inoculum per 100 ml of thioglycollate broth.
atures indicated. Unless otherwise stated, freshly infected feed was provided ad libitum daily for five days. Water was always available free choice.
NUMBER OF C. perfringens The numbers of C. penfringens per ml of thioglycollate broth prior to its addition to feed and the numbers of these organisms per gram of inoculated and incubated feed just before feeding were determined as follows. One ml of broth was added to 9 ml of phosphate buffered saline (PBS), then mixed and four 0.2 ml volumes of appropriate dilutions were placed on blood agar plates. One gram of feed was added to 99 ml of PBS, mixed, diluted and plated as above. All plates were incubated anaerobically overnight and the mean colony count per ml was determined. CHICKENS
FEED
Commercial chick starter6 was used throughout except for one experiment in which a laboratory prepared feed7 was used. This latter feed, in sealed preserving cans, was sterilized with 5 Mrads of gamma irradiation8. INOCULATION OF FEED
When broth cultures were added, weighed amounts of feed were prepared and broth was mixed in such a way as to provide an even distribution of the broth throughout the feed. The feed/broth ratio in this mixture was 1/1.25 (w/v) and when incubated this was done aerobically at the temper-
3Difco Laboratories, Detroit, Michigan, U.S.A. 4War-ner-Chilcott Laboratories, Toronto, Ontario. 5Thioglycollate Broth Brewer's modified, BBL, Division of Becton, Dickinson, Clarkson, Ontario. 6Master Feed Krums, Maple Leaf Mills Limited, Toronto, Ontario. IPrepared by Department of Animal and Poultry Science, University of Guelph. SAtomic Energy of Canada, Limited. Ottawa, Ontario.
54
Columbian rock chicks were provided by the Department of Animal and Poultry Science from a small flock maintained for experimental purposes. Chicks were started in a Petersime' battery brooder in an isolation room in the isolation facilities of the Department of Veterinary Microbiology and Immunology. They were transferred to heated brooders in another room in groups of ten to 25 birds for experimentation at two to four weeks of age. A group of control birds equal to at least 10% of the birds on test was maintained on normal feed and water either in the test room or in a separate room. Necropsies were performed on all dead birds. Gross lesions in the intestine were recorded and representative sections of affected areas were collected for histological examination and bacteriological culture.
RESULTS The isolates obtained and results of the tests to determine their toxin are shown in Table I. This table shows that type A toxin 9Petersime Incubator Company, Gettysbury, Ohio, U.S.A.
Can. J. comp. Med.
TABLE I. Toxin Production from Field Isolates of C. perfringens Isolate
2153
.............
74-379 . ......................... 74-590 . ......................... L45 .. 2934 .......................... 2945 ..... ......... -NT = No toxin detected ',ND Not done
Toxin NT-
NDb A A NT A
could be demonstrated from only three of the five strains tested. Initial experiments were carried out with groups of ten birds each. The cells recovered by centrifugation from 600 ml of a 15 hr thioglycollate culture of isolate 2153 were resuspended to 50 ml in phosphate buffered saline pH 7.0 and 1 ml amounts were inoculated into the crop by means of an 18 gauge teat cannula. Four doses were given at four hour intervals for one day. No deaths resulted and the intestines were normal when examined macroscopically three days postinoculation. The next experiment involved the use of a 24 hr thioglycollate broth culture of 2153 and feed containing 5 or 20% starch. Approximately 750 ml of broth culture was mixed with 600 g of feed and incubated aerobically for 24 hours. A like amount of feed was inoculated and incubated anaerobically. This feed was provided as the only feed to two week old chicks till consumed. The results as presented in Table II suggested that the addition of starch might be important in disease reproduction and a further experiment was done with varied levels of starch in the ration. Two hundred grams of feed was mixed with 250 ml of a 24 hr broth culture of 2153 and the mixture was incubated aerobically for 24 hrs. This was repeated for a total of four daily feedings. The results presented in Table III suggested that a level of 2% starch was the most satisfactory. Further tests were done with differing levels of starch with varied results, generally lesions and mortality were greatest with 2% starch and all further trials, except as indicated, were done with 2% starch in the feed. To test the effect of incubation time and temperature of broth and broth feed mixtures, broths were incubated for nine, 15 or 24 hrs at 33, 37 or 41°C and the broth feed mixtures were incubated for nine or
Volume 40
January, 1976
Fig. 1. Broiler intestine from a case of experimentally reproduced necrotic enteritis with a typical diphtheritic membrane.
15 hrs at these same temperatures. The birds were fed daily for five days. The results as presented in Table IV suggested that the most favourable results were obtained when the broth was incubated for 15 hrs at 37°C and the broth feed mixture was incubated for nine hrs at 33°C and this was adopted as a standard incubation time and temperature except as indicated. In an attempt to determine whether greater mortality might result if the test strains were freshly isolated and used for the next experiment, several tests were carried out. These were done to compare initial isolates from different field cases using the standard incubation conditions developed and further to test reisolates as designated by RI, etc. In addition, feed sterilized by irradiation was inoculated and incubated to determine whether similar results might be obtained. Also, to determine whether the medium
55
TABLE II. Comparison of Aerobic and Anaerobic Incubation of Broth Feed Mixture for Reproduction of Necrotic Enteritis % Starch Dead/Treated
Incubation
Aerobic
5
............
Aerobic .20 Anaerobic .20 -NE = Necrotic enteritis TABLE III. Effect of Varied Levels of Starch Mixed with Feed on the Reproduction of Necrotic Enteritis
% Starch
Dead/Treated
0.............. 1............ 2............ 5............ -NE = Necrotic enteritis
1/10 0/10 4/10 0/10
Remarks NE-
NE
influenced the results obtained, a series of birds received feed inoculated with thioglycollate broth U.S.P.10 The results as presented in Table V suggest that virulence may be increased by bird passage and reisolation. No further attempts were made with isolate 74-379 because of the low mortality produced. Similar results had occurred with isolates 2934 and 2945. Thioglycollate broth U.S.P. appeared to be as satisfactory as Brewer's modified for growth of the test strain. To test the prophylactic effect of penicillin and chloramphenicol" upon necrotic enteritis, groups of birds received L45-R2 infected feed prepared as above and were provided with water containing 100,000 I.U. penicillin/litre or 110 mg chloramphenicol/litre. Treated water was provided eight hours prior to infection exposure and continued for six days following the last feeding of infected feed. Fresh antibiotic containing water was provided every 12 hours throughout the experimental period. The results as shown in Table VI indicate that penicillin at the test level was effective in preventing mortality while chloramphenicol reduced and delayed mortality but did not prevent the disease. A further experiment was carried out to assess the effect of feed with and without starch using the standard incubation times
0OBBL, Thioglycollage broth U.S.P.
1Francomycin 125, P.V.U. Inc., Hamilton, Ontario.
56
2/10
3/10 2/10
Remarks
Lesions of NE-
Lesion suggestive of NE Intestine grossly normal
and also to compare the standard incubation of the broth feed mixture with unincubated inoculated feed and further to compare feeding for one, two or the usual five days. Isolate L45-R3 was used for this test and the broth was incubated for 15 hrs at 370C. The results as shown in Table VII indicate that a longer exposure to infection results in higher mortality. It does not appear that starch is necessary and furthermore incubation of the broth feed mixture did not produce as high mortality as did the inoculated feed that was not incubated.
Fig. 2. An early lesion of necrotic enteritis with necrosis in the lamina propria. Apex of villus at right. H and E. X400.
Can. J. comp. Med.
TABLE IV. Effect of Varied Incubation Time and Temperature on the Production of Necrotic Enteritis Using Strain 2153 Broth Incubation Time/hr Temp/C°
Feed Incubation Temp/C° Time/hr
33 9 33 33 15 33 33 37 15 33 24 33 37 9 37 37 37 15 37 33 15 24 37 37 41 9 41 24 41 41 -All dead birds had lesions typical of necrotic enteritis
9 9 9 15 15 9 9 15 15 15
Results
-Dead/Treated 0/20 3/20 2/20 3/20 4/40 1/20 6/20 2/40 1/20
4/20
TABLE V. Pathogenicity Tests of Various Isolates and the Effect of Reisolation
Isolates Dead/Treated % Mortality 6.9 2153R3....... 11/16Cc 13.7 2153R4 22/160 13.1 2153R5....... 21/160 1.0 74-379 1/100 9.0 74-590 9/100 17.0 74-590R1 17/100 19.0 L45 ......... 19/100 9.0 L458 ......... 9/100 26.0 L45R1....... 26/100 28.0 L45RIb 17/60 WFeed sterilized by Gamma irradiation bGrown in thioglycollate broth, U.S.P.. BBL All dead birds had lesions of NE .......
........
.......
of C. perfringens. Histological sections from intestinal lesions of birds infected with various isolates showed early (Fig. 2) and advanced lesions (Fig. 3) of NE. Representative colony counts are presented in Table VIII where it may be seen that although there were variations in the incubation times the differences in counts from broth cultures of the four strains shown does not appear to be great. Similarly with the counts from the feed, on occasion almost identical counts were obtained with three of the four strains used, although the variation between samples was found from Fig. 3. Necrotic enteritis with necrosis of all villus somewhat greater than those structures. H and E. X40. broth. It is also apparent that the counts from inoculated feed were markedly less than those from broth and much less than With the exception of two birds (Table can be accounted for on the basis of diluII) which died in early experiments, macro- tion by feed. Although not evident from scopic lesions typical of necrotic enteritis the table there did not appear to be a rela(Fig. 1) were observed in the intestine of tionship between the counts and the actiall birds which died following experimental vity obtained with respect to disease reinfection. Gram stain smears and cultures production for any particular inoculated from these lesions showed large numbers feed.
Volume 40
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January, 1976
57
DISCUSSION
The intestinal lesions in the experimentally reproduced disease were identical macroscopically and microscopically to those observed in field cases (2, 5, 7). Occasionally deaths occurred within 24 hours after consuming inoculated feed but deaths were more common after 36 hours with the peak mortality being reached at 48 hours. Deaths continued to occur throughout the five day experimental period. In later experiments when birds were kept for five days beyond the usual five day
test period it was observed that a few deaths occurred for the next three days. The acute nature of the disease was often seen with sick birds dying within 30 minutes after illness was observed. The finding that exposure to C. perfringens for a 24 hour feeding period resulted in 12% mortality while a five day feeding period resulted in mortality as high as 26% suggests a relationship between continued exposure to the organisms and disease incidence (Table VII). However, since 26% mortality was the maximum achieved in these studies it is apparent that other factors, as yet undetermined, may be important in reproducing
TABLE VI. Effect of Penicillin and Chloramphenicol in the Prevention of Mortality Due to Necrotic Enteritis
Treated Controls %0 Antibiotic Trial Dead/Treated Dead/Treated % Mortality 1 Penicitlin ............. 0 17 0/100 17/100 Penicillin ............. 2 0 21 0/90 19/90 Chloramphenical ....... 1 12a 12/100 25 25/100 *First deaths occurred 48 hours after deaths had occurred in control birds TABLE VII. Comparison of Incubation Time. Level of Starch and Exposure Periods for the Production of Necrotic Enteritis
Incubation Temp(C°)/Time(hrs) 33 9 33 9 33 9 33 9 33 9 0 0
%0 Starch 2 2 2
0 0 0
No. Feedings (days) 1 2 5 5 5 5
% Mortality 12 11 17 14 19 26
Dead/Treated 11/92
10/92
17/100 14/100 19/100 26/100
TABLE VIII. Colony Counts of Four Cultures of Clostridium perfringens from Broth and Inoculated Feed
Broth
Isolates 2934
2153 L45 590
Hrs. Incub.9
Count/ml X
107 44
8
18
15 15 15 15 15
18 27 15 28 20
Feed
Hrs. Incub. 5 5 5 5 15 15 9 9 9 9 9
Tenirp.
Incub. 37 37 37 37 37 37 33 33 33 33 33
c7^
Starch 0 1 2 3 2 4
2
2 0 2 2
Count/g X
107 7.3 5.8 2.9 4.5 2.6 1.9 2.7 5.0 96 8.5
4.6
Incubated at 370C
58
Can. J. comp. Med.
the disease. In preliminary studies starch appeared to be useful in assisting in disease reproduction and it was used throughout most of these studies. In a few final experiments (Table VII) where it was omitted the results suggest that it was not necessary. The role of such additives can only be determined with a series of experiments comparing diets with and without such substances. This disease appears to be one in which if the disease is manifest the bird dies, since an examination of survivors indicated that the intestines of such birds were grossly normal, while organisms resembl-
ing C. perfringens could be readily found throughout the intestinal tract. It is postu-
lated that multiplication of C. perfringe'ns
to threshold numbers within a particular time period is one of the major requirements for disease to occur. Whether continued feeding of infected feed beyond the tested five day period would assist or whether the critical time is actually within this period remains to be determined. At any rate, the percent mortality in these studies does exceed those reported from the field (5, 6) and it is likely that the level and duration of exposure are important here. It is possible that with a prolonged low level exposure sufficient immunity could develop to cause the disease to be selflimiting within a flock. Although the pathogenesis of intestinal lesions has not been worked out a possible pathogenesis has been proposed (7). From these studies it appears that oral feeding
with a virulent strain of C. perft ingens will
allow the disease to be consistently reproduced. With this method of reproduction the exact mechanism of lesion production can be studied. From this (Table I) and previous studies (7) it seems that all iso-
lates from necrotic enteritis are C. perfrin-
gens type A or do not yield enough toxin in vitro to permit typing. Such isolates are considered to be type A (12). Penicillin added to the drinking water (Table VI) protected birds completely and
Volume 40 - January, 1976
this antibiotic is commonly used in Ontario to treat field cases. Chloramphenicol, at the level used, delayed and reduced but did not prevent mortality (Table VI). These findings may be explained on the basis that the action of chloramphenicol may have been incomplete in the intestine. However, all strains studied were susceptible in vitro to both antibiotics using the disc method.
ACKNOWLEDGMENTS The authors thank Dr. J. R. Pettit for his assistance with the microscopic examination of intestinal lesions and Dr. N. C. Palmer for help with photography. REFERENCES 1. BERNIER, G. and R. FILION. Necrotic enteiritis in br oiler chickens. J. Am. vet. med. Ass. 158: 18961897. 1971. 2. BERNIER, G., J. B. PHANEUF et R. FILION. Ent& rite necrotique chez le poulet de gril I. Aspects clinico-pathologique. Can. J. comp. Med. 38: 280285. 1974. 3. BERNIER, G., R. FILION, R. MALO et J. B. PHANEUF. Enterite necrotique chez le poulet de gril II. Caracteres des souches de Clostridium perfringens isolees. Can. J. comp. Med. 38: 286-291. 1974. 4. DAVIS, R. B., J. BROWN and D. L. DAWE. Quail -biological indicators in the differentiation of ulcerative and necrotic enteritis of chickens. Poult. Sci. 50: 737-740. 1971. 5. HELMBOLDT, C. F. and E. S. BRYANT. The pathology of necrotic enteritis in domestic fowl. Avian Dis. 15: 775-780. 1971. 6. LONG, J. R. Necrotic enteritis in broiler chickens I. A review of the literature and the prevalence of tne disease in Ontario. Can. J. comp. Med. 37: 302308. 1973. 7. LONG, J. R., J. R. PETTIT and D. A. BARNUM. Necrotic enteritis in broiler chickens II. Pathology and proposed pathogenesis. Can. J. comp. Med. 38: 467-474. 1974. 8. NAIRN, M. E. and V. W. BAMFORD. Necrotic enteritis of broiler chickens in western Australia. Aust. vet. J. 43: 49-54. 1967. 1. PARISH, W. E. Necrotic enteritis in the fowl. 1. Histopathology of the disease and isolation of a strain of Clostridium welchii. J. comp. Path. 71: 377-393. 1961. 10. PARISH, W. E. Necrotic enteritis in the fowl. II. Examination of the causal Clostridium welchii. J. comp. Path. 71: 394-404. 1961. 11. PARISH, W. E. Necrotic enteritis in the fowl. IHI. The experimental disease. J. comp. Path. 71: 405413. 1961. 12. SMITH. L. DS. Clostridia. In Manual of Clinical Microbiology. Eldited by J. E. Blair, E. H. Lennette and J. P. Truant. pp. 280-283. Bethesda, Maryland: American Society of Microbiology. 1970.
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