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Colonization of the Intestinal Tract by Clostridium Perfringens and Fecal Shedding in Diet-Stressed and Unstressed Broiler Chickens
Colonization of the Intestinal Tract by Clostridium perfringens and Fecal Shedding in Diet-Stressed and Unstressed Broiler Chickens S. E. Craven Poultry Microbiological Safety Research Unit, USDA, Agriculture Research Service, R. B. Russell Agricultural Research Center, PO Box 5677, Athens, Georgia 30604-5677 on each of the two diets that were sampled from 2 to 21 d after gavage, C. perfringens was recovered more frequently from the crop, proventriculus, duodenum, jejunum, ileum, ceca, and feces, but not the gizzard, of birds on the 50% rye diet as compared to those on the cornbased diet. From 2 to 21 d, the intestinal numbers of C. perfringens in contaminated birds on the corn-based diet were ≤log10 6.2 and did not increase in any portion of the intestinal tract. Numbers of C. perfringens during this time period increased 3.3 to 4.9 log factors in the ileum, ceca, and feces of birds on the 50% rye diet to as high as log10 7.1 to 7.9 at 21 d. This study confirms that addition of rye to the diet of chickens can increase the numbers of C. perfringens in the ceca of broiler chickens and extends these findings to demonstrate increases in the numbers and frequency of recovery of C. perfringens in other parts of the intestinal tract.
(Key words: Clostridium perfringens, broiler chicken, intestine, rye) 2000 Poultry Science 79:843–849
poultry flocks worldwide that suffer from necrotic enteritis (Parrish, 1961; Long et al., 1974; Tsai and Tung, 1981). A better understanding of conditions that favor the proliferation of C. perfringens is important in determining the cause and spread of necrotic enteritis (Paulus and Ruckebusch, 1996). Factors that predispose flocks to necrotic enteritis include management stress, changes in dietary formulation, alteration of feeding programs, and subclinical intestinal coccidiosis (Shane et al., 1985). Grains such as wheat, barley, or rye, when added to a corn-based feed have been shown to inhibit weight gain, increase C. perfringens numbers, induce necrotic enteritis, and increase bird mortality (Misir and Marquardt, 1984; Riddell and Kong, 1992; Takeda et al., 1995). Little information is available about the change in C. perfringens numbers in birds before overt symptoms of the disease are observed, the relative numbers recovered from certain parts of the intestinal tract, or the roles of diet and fecal shedding in the spread of the disease. The current study was conducted to determine the rate and extent of increase in the intestinal population of C. perfringens, the numbers shed in fecal droppings, and the relationship of fecal numbers to numbers found in the
INTRODUCTION Clostridium perfringens is recognized as an enteric pathogen in humans, domestic animals, and wildlife worldwide (Songer, 1996). Necrotic enteritis, a disease of poultry caused by this organism, leads to the development of necrotic lesions in the gut wall, as well as increased mortality. Clostridium perfringens is also associated with other poultry diseases, including avian malignant disease, also known as gas edema disease (Hofacre et al., 1986), gizzard erosions (Fossum et al., 1988), and gangrenous dermatitis (Gerdon, 1973). It has been implicated in the growth depression of poultry (Stutz and Lawton, 1984) and the condemnation at slaughter due to liver abnormalities (Hutchison and Riddel, 1990; Onderka et al., 1990). A high number of C. perfringens in the intestinal tract and associated necrotic lesions have been detected in
Received for publication June 30, 1999. Accepted for publication February 23, 2000. 1 To whom correspondenceshould be addressed: scraven@asrr. arsusda.gov.
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ABSTRACT Commercial broiler chicks were given a three-strain composite of bacitracin-resistant Clostridium perfringens by oral gavage and were sampled periodically to determine the dynamics of C. perfringens colonization of the intestinal tract of broiler chickens and fecal shedding. After gavage, the chicks were divided into two groups and placed in isolators, one group received a traditional corn-based diet, and the other group received the same diet supplemented with 50% rye to place the birds under dietary stress. The numbers of bacitracin-resistant C. perfringens in various parts of the intestinal tract, liver, and feces were determined using a selective plating medium containing bacitracin. In chickens on the corn-based or 50% rye diet, C. perfringens was isolated infrequently from the various parts of the intestinal tract, liver, or fecal droppings during the first 36 h following the last gavage. For the 24 chickens
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intestinal tract during the first 3 wk of grow out. Experimental birds were given either a corn-based diet to simulate standard commercial conditions or a diet supplemented with 50% rye to expose the birds to dietary stress.
MATERIALS AND METHODS Experimental Design
Chickens Broiler chicks were housed in sanitized and temperature-controlled isolator units with wire mesh floors and positive air pressure for the duration of the experiment. Water was provided ad libitum through nipple waterers.
Feed Two types of feed were used: 1) a corn-based, unmedicated mash feed that contained 57.3% ground corn, 33.5% dehulled soybean meal, 3.2% poultry fat, and 3.0% poultry by-product meal with the remainder consisting of defluorinated phosphate, limestone, vitamin mix, salt, D,L-methionine, and trace minerals2 and 2) the cornbased feed supplemented with 50% (wt/wt) organic rye flakes.3 No attempts were made to equalize nutritional or caloric values for the two feed types. Feed was provided ad libitum for consumption throughout the trials.
2 Poultry Science Department, University of Georgia, Athens, GA 30602. 3 Arrowhead Mills, Hereford, TX 79045. 4 Difco Labs, Detroit, MI 48232. 5 Tekmar Co., Cincinnati, OH 45249. 6 Unipath, Co., Oxoid Division, Ogdensburg, NY 13669.
Clostridium perfringens 5227, 5229, and 5230, bacitracinresistant strains previously isolated from the outbreaks of necrotic enteritis, were obtained from S. Thayer, Poultry Disease Research Center, the University of Georgia, Athens, Georgia. The strains were inoculated from stock cultures into cooked meat medium4 and incubated at 37 C for 24 h. Equal volumes from the liquid portions of each of the three cultures were combined. From the composite culture, 0.1 mL (approximately 108 cells) was given to each chick in the C. perfringens-treated group by oral gavage on the day of hatch. A second dose of 0.1 mL of a fresh composite culture was given on the following day to each of these chicks.
Bacteriological Sampling At given intervals after the oral gavage with C. perfringens or saline (12, 24, and 36 h in Trial 1 and 2, 7, 14, and 21 d in Trials 1 and 2), birds were euthanatized by cervical dislocation to sample their intestinal tract. A 5 cm length of each intestinal sampling location was excised with a pair of surgical scissors, placed into a stomacher bag with sterile buffered peptone-water to give an initial 1/5 (wt/vol) dilution, and cut to allow homogenization of contents. The locations in the intestinal tract that were sampled included the crop, proventriculus, gizzard, duodenum, ileum, jejunum, and cecum. The intestinal segments and their contents were homogenized using the Stomacher Lab-blender 80.5 Serial dilutions were made in sterile saline. The liver and fresh fecal droppings were sampled in a similar manner. After stomaching, dilutions were prepared, and 0.1 mL of each dilution was placed into petri dishes and mixed with the selective plating medium consisting of Perfringens Agar Base6 with added egg yolk suspension, 20 µg/mL kanamycin, and 20 µg/mL bacitracin. Agar plates were overlaid with the same plating medium but without egg yolk. After anaerobic incubation for 48 h at 37 C, black colonies with white halos, which were indicative of lecithinase activity, were counted as C. perfringens. In a preliminary study conducted to determine the effectiveness of the recovery medium for enumerating bacitracin-resistant C. perfringens in intestinal contents, cecal contents from unchallenged 3-wk-old chickens were inoculated with the C. perfringens composite, diluted, and plated on the recovery medium containing egg yolk, kanamycin, and bacitracin. An uninoculated portion was plated as a control. Lecithinase-positive and -negative colonies were picked and confirmed according to Harmon (1984). Isolates that were lecithinase positive, displayed motility, reduced nitrate to nitrite, produced acid and gas from lactose, and liquified gelatin within 48 h were considered C. perfringens. Fifty colonies of sulfite-reducing bacteria (black) demonstrating a lecithinase reaction (white halo) and fifty colonies of sulfite-reducing bacteria with no lecithinase reaction were characterized (data not shown). None of the black, lecithinase-negative colonies
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In each of two trials, 120 day-of-hatch chicks obtained from a local broiler chicken hatchery were divided into four equal groups, and each group was placed in a separate isolator. Chicks in two isolators were maintained on corn-based feed, and chicks in the other two isolators were on 50% (wt/wt) rye feed. For each feed treatment, chicks in one of the two isolators were given C. perfringens, and the chicks in the other isolator were given a saline control. In Trial 1, chickens were euthanatized at 12, 24, or 36 h or at 2, 7, 14, or 21 d after C. perfringens administration and intestinal samples were prepared to determine counts of bacitracin-resistant C. perfringens with a selective plating medium. In Trial 2, chickens were not sampled at 12, 24, or 36 h but were sampled as in Trial 1 at 2, 7, 14, and 21 d. At 12 h in Trial 1 and 21 d in Trials 1 and 2, control chicks receiving no C. perfringens were also sampled and plated in the same manner. Three birds per sampling time per diet per trial were sampled for a total of 66 birds. More were not used because previous studies have shown that the numbers of C. perfringens were higher in chickens on a diet containing rye compared to numbers in chickens on a corn-based diet.
Clostridium perfringens Strains
CLOSTRIDIUM PERFRINGENS INTESTINAL COLONIZATION
were confirmed as C. perfringens. Of those colonies, 65% were facultative anaerobes, and 35% were strict anaerobes. All of the black, lecithinase-positive colonies were determined to be strict anaerobes. Of those, 86% were confirmed as C. perfringens. Some of the remaining 14% were likely false negatives because black, lecithinase-positive colonies did not appear on plates of similar dilutions of a cecal sample not inoculated with C. perfringens (data not shown). When a culture of the three-strain composite of C. perfringens without cecal contents was plated on the recovery agar containing kanamycin and bacitracin, numbers were similar to those on basal medium without antibiotics (data not shown). The results indicated that the recovery medium was selective for and not inhibitory to the experimental strains of C. perfringens.
Statistical Analysis
RESULTS At 12, 24, and 36 h after the chickens were gavaged with the experimental three-strain composite of C. perfringens, few C. perfringens were recovered from the intestinal tracts of birds fed the corn-based feed (Table 1). Only the crop and ileum of one bird tested positive for C. perfringens. C. perfringens was recovered occasionally during the first 36 h from chickens on 50% rye feed and in more birds and in more intestinal sampling locations than for birds on corn-based feed (Table 1). The numbers of C. perfringens from any portion of the intestinal tract at 12 to 36 h were ≤log10 5.5/g of intestinal sample from birds on either of the two diets. The frequency of C. perfringens recovered from most parts of the intestinal tract decreased between 12 and 36 h regardless of the diet. C. perfringens was not recovered from the ceca of control birds. For the four sampling periods of 2, 7, 14, and 21 d postchallenge, C. perfringens was recovered on at least two of the four sampling periods from each portion of the intestinal tract of some of the birds challenged with the three-strain composite and subsisting on corn-based feed (Table 2). None were detected in control ceca. The
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Dataxiom Software Inc., Los Angeles, CA 90010.
highest frequency of isolation was found at Day 7 in the crop (six of six birds) and ileum (five of six birds). On Day 7 C. perfringens was found less frequently in the proventriculus, gizzard, duodenum, jejunum, and ceca and on Days 2, 14, and 21 for the crop and ileum. In those intestinal sampling locations where two to six birds were positive for C. perfringens, mean C. perfringens numbers for those birds on a given day from 2 to 21 d were similar in the different locations of the intestinal tract sampled. An exception was Day 14 on which fecal counts were significantly higher than ileal counts. No significant increases in mean C. perfringens numbers were observed in C. perfringens-positive birds on a corn-based diet during the period of 2 to 21 d. No consistent increase in isolation rates were observed, only a transient increase in recovery of C. perfringens from the crop, ileum, and feces on Day 7. In the rye-fed birds, all intestinal sampling locations of one or more birds contained C. perfringens on Day 2 (Table 3). By Day 7, C. perfringens could be found in the crop and ileum of all six chickens sampled and in all intestinal sampling locations of two of the six birds. Results were similar to those observed for corn-fed birds at Day 7 (Table 2), in that the highest frequency of recovery of C. perfringens occurred in the crop and the ileum. From 7 to 21 d, the frequency of isolation from the crop decreased for birds on the rye-supplemented diet, but the frequency of isolation from other intestinal sampling sites remained the same or increased. In contrast to the C. perfringens counts from positive birds on corn-based feed, the mean numbers of C. perfringens in positive birds on rye-supplemented feed varied in different parts of the intestinal tract on Days 7, 14, and 21. Mean numbers in positive birds increased from 2 to 21 d in the ileum and ceca by as much as 3.3 to 4.9 log factors with mean counts in birds at 21 d ranging from log10 6.8 to 7.9/g in these intestinal locations. The numbers of C. perfringens detected in the fecal droppings of birds generally reflected the numbers found in the ileum. In chickens on the corn-based and 50% rye diets, C. perfringens was not detected in the feces at 12 to 36 h (Table 1). On 2, 7, 14, and 21 d, the frequency of isolation from feces of chickens on a corn-based diet was 0 to 3 out of 6 sampled birds with numbers ≤ log10 6.2/ g for positive samples. The frequency of isolation from samples of the ileum was one to five of six birds with numbers ≤ log10 4.3/g (Table 2). The frequency of isolation from the feces of birds on the 50% rye diet was four to five out of six sampled birds with numbers ranging from log10 3.8 to 7.1/g for C. perfringens-positive samples compared to an isolation frequency of two to six out of six in the ileum with mean numbers ranging from log10 3.7 to 7.5 in positive samples (Table 3). In this study, the frequency of isolation of C. perfringens from the liver of chickens on either diet was low (Tables 1 to 4). The numbers found in C. perfringens-positive livers were also low. A comparison of the occurrence of C. perfringens in chickens on a corn-based diet (Tables 1 and 2) and on a 50% rye diet (Tables 1 and 3) indicated that mean numbers in positive birds were higher throughout most of the
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Statistical analyses were performed using Statistica for Windows software.7 Student’s t-test was used to determine significant differences for C. perfringens numbers between different intestinal sampling locations and different sampling days. Significant differences in the frequency of recovery of C. perfringens from intestinal samples of chickens were determined by pooling the results from Days 2, 7, 14, and 21 for each intestinal sampling location of birds on a given diet. Fisher’s exact test was applied to the pooled data that had been organized into 2 × 2 contingency tables. Significance for both tests was determined at P ≤ 0.05.
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CRAVEN TABLE 1. Occurrence of Clostridium perfringens in chickens at 12, 24, and 36 h after final gavage and when fed a corn-based or 50% rye diet in Trial 1 Chickens1 positive for C, perfringens Intestinal sampling location
Corn-based diet 12 h
Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Ceca control3 Feces Liver
2
1 (5.5) 0 0 0 0 1 (2.7) 0 0 0 0
50% rye diet
24 h
36 h
12 h
0 0 0 0 0 0 0 ND4 0 0
0 0 0 0 0 0 0 ND 0 0
2 0 2 0 1 1 1 0 0 1
(3.3 ± 0.8) (2.9) (2.3) (2.5) (5.5) (3.3)
24 h
36 h
0 0 0 0 1 (2.9) 0 1 (4.6) ND 0 0
0 1 (5.3) 0 1 (4.8) 0 0 0 ND 0 1 (4.2)
1
Out of three chickens per diet per day. Values in parentheses = mean log10 C. perfringens colony-forming units per gram of sample material from C. perfringens-positive chickens ± SD. 3 Cecal samples from chickens housed in separate isolators given saline but not the three-strain composite of C. perfringens. 4 Not determined. 2
numbers at 2 d, the high numbers at 21 d, and the numbers from Days 7 and 14 for the rye-fed birds.
DISCUSSION Addition of rye to the diet of chickens has been reported to increase numbers of C. perfringens in the cecal contents (Takeda et al., 1995). Our current study confirms and extends these findings by demonstrating an increase in numbers of C. perfringens from 2 to 21 d of age in the ileum, ceca, and feces of birds given a diet containing 50% rye. Also, a higher frequency of recovery through most of the intestinal tract and higher numbers in the jejunum, ileum, and ceca of C. perfringens-positive birds occurred as compared to corresponding numbers in birds on a corn-based diet.
TABLE 2. Occurrence of Clostridium perfringens in chickens at 2, 7, 14, and 21 d after final gavage and when fed a corn-based diet in Trials 1 and 2 Intestinal sampling location Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Ceca control3 Feces Liver
Chickens1 positive for C. perfringens 2d
7d
0 2 (4.5 ± 2 (3.4 ± 2 (6.0 ± 0 1 (2.8) 1 (3.4) ND4 0 2 (3.9 ±
6 (3.9 ± 1.1) 0 1 (3.5) 0 1 (2.3) 5 (2.9 ± 0.7)a 1 (3.9) ND 3 (4.9 ± 1.7)a 0
a,2
0.8)a 1.9)a 1.0)a
1.1)a
14 d
21 d
1 (4.0) 1 (2.7) 1 (2.0) 1 (3.7) 1 (3.5) 2 (3.0)b 0 ND 2 (4.5 ± 0.2)a 0
0 0 2 1 2 3 2 0 1 0
(3.1 ± (2.0) (3.0 ± (4.3 ± (4.9 ±
0.3)a 1.4)a 1.2)a 2.7)a
(6.2)
a Means of intestinal and fecal samples within columns with no common lowercase superscripts differ significantly (P ≤ 0.05). 1 Out of six chickens, three chickens per trial per day. 2 Values in parentheses = mean log10 C. perfringens colony-forming units per gram of sampled material from C. perfringens-positive chickens ± SD. 3 Cecal samples from chickens housed in separate isolators and given saline but not the three-strain composite of C. perfringens. 4 Not determined.
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intestinal tract for chickens on the rye-supplemented diet and suggested that recovery rates were also higher. A direct comparison of birds on the two diets with data pooled from Days 2, 7, 14, and 21 is shown in Table 4. Of the 24 birds on each of the diets sampled, the recovery of C. perfringens was significantly higher in the crop, proventriculus, duodenum, jejunum, ileum, ceca, and feces of birds on the 50% rye diet. Recovery rates from the gizzard and liver were not significantly different in birds on the two diets. The overall numbers (mean log10 cfu/ g of intestinal material from all positive birds) were significantly higher (by 2.6 log factors) in the jejunum of birds given 50% rye. The overall numbers in the ileum and ceca were 2.7 to 2.8 log factors higher in birds on the rye diet, but this difference was not significant due to the large variation that resulted by averaging the low
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CLOSTRIDIUM PERFRINGENS INTESTINAL COLONIZATION TABLE 3. Occurrence of Clostridium perfringens in chickens at 2, 7, 14, and 21 d after final gavage and when fed a 50% rye diet in Trials 1 and 2 Intestinal sampling location Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Ceca control3 Feces Liver
Chickens1 positive for C. perfringens 2d
7d 2
1 (4.8) 4 (4.3 ± 0.7)ab,x 2 (4.6 ± 0.6)a,x 4 (4.4 ± 1.5)ab,x 3 (3.9 ± 1.6)ab,x 2 (3.7)b,y 1 (3.0) ND4 4 (3.8 ± 0.9)ab,y 0
14 d
6 (3.5 ± 2 (4.5 ± 2 (3.4 ± 4 (5.1 ± 5 (4.6 ± 6 (4.9 ± 3 (6.9 ± ND 5 (5.0 ± 2 (3.2)
bcx,2
1.8) 2.7)abc,x 0.5)c,y 1.3)abc,x 1.7)abc,x 2.2)abc,xy 0.5)a,y 0.5)b,x
5 (4.2 4 (4.0 3 (5.0 4 (5.2 6 (5.7 5 (6.7 5 (6.5 ND 5 (6.2 0
21 d ± ± ± ± ± ± ±
c,x
1.5) 1.4)c,x 1.7)abc,xy 2.0)abc,x 1.0)abc,xy 0.8)a,x 0.5)ab,y
± 1.5)abc,xy
2 3 5 5 5 6 6 0 5 2
(4.8 (4.5 (4.0 (5.0 (6.8 (7.5 (7.9
± ± ± ± ± ± ±
0.3)c,x 2.4)bc,x 1.7)c,xy 2.2)abc,x 0.7)ab,x 1.0)ab,x 0.7)a,x
(7.1 ± 1.8)ab,x (3.4 ± 0.9)
a,b
Clostridium perfringens is commonly found in the intestinal tract of poultry (Tschirdewahn et al., 1991), but the occurrence of the poultry disease, necrotic enteritis, is sporadic (Cowen et al., 1987). A complete understanding of the factors contributing to infection of the intestinal tract and subsequent events leading to necrosis is lacking (Truscott and Al-Sheikhly, 1977). Examination of the intestinal tract of affected birds in flocks with this disease and in controlled studies shows that high numbers of C. perfringens cells are present and are intimately associated with damaged tissue. Because these high numbers are not generally found in the intestinal tract of birds from healthy flocks, conditions favoring proliferation of C. perfringens in the intestinal tract appear to be essential for disease outbreaks. In evaluating increases in intestinal numbers of C. perfringens and effects of independent variables in this study,
numbers in C. perfringens-positive birds only were considered because the value for negative birds would skew the means, increase the variability among means, and perhaps mask changes or differences in C. perfringens numbers in that portion of the bird population that was contaminated by this organism. An analysis of C. perfringens numbers in positive birds by Student’s t-test and frequency of recovery of C. perfringens by Fisher’s exact test indicated that the intestinal sampling location, diet, and day of sampling could influence the occurrence of C. perfringens (P ≤ 0.05). In contrast, when an ANOVA of the total bird population (C. perfringens-positive and negative birds) was conducted, the intestinal sampling location (but not the sampling day, diet, or trial number) was shown to have a significant effect on C. perfringens numbers (P ≤ 0.05) (data not shown). However, there were interactions between sampling day and intestinal
TABLE 4. Occurrence of Closridium perfringens in chickens from Trials 1 and 2 pooled for sampling days 2, 7, 14, and 21 d. Chickens were maintained ad libitum on a corn-based or 50% rye diet Intestinal sampling location Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Feces Liver
Recovery of C. perfringens1 Corn-based diet 7 3 6 4 4 11 4 6 2
50% rye diet a
14 13a 12 17a 19a 19a 15a 19a 4
Mean cfu/g of intestinal material2 Corn-based diet 3.9 3.9 3.1 4.2 3.0 3.3 4.2 5.0 3.9
± ± ± ± ± ± ± ± ±
1.0 1.2 1.0 1.8 1.0 0.9 1.5 1.2 1.1
50% rye diet 4.0 4.3 4.4 4.9 5.6 6.1 6.9 6.1 3.0
± ± ± ± ± ± ± ± ±
1.2 1.5 1.5 1.7 1.4a 2.0 1.5 1.5 0.9
a Superscripts indicate a significant difference between chickens on the different diets for the recovery of C. perfringens or mean colony-forming units of C. perfringens per gram (P ≤ 0.05). 1 Number of chickens positive for C. perfringens at a given intestinal location of the 24 total chickens on a given diet and sampled from 2 to 21 d. 2 Mean log10 C. perfringens colony-forming units per gram of material sampled at a given intestinal location for the positive birds of the 24 chickens on a given diet and sampled from 2 to 21 d.
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Means of intestinal and fecal samples within columns with no common superscript differ significantly (P ≤ 0.05). x,y Means within rows with no common superscript differ significantly (P ≤ 0.05). 1 Out of six chickens, three chickens per trial per day. 2 Values in parentheses = mean log10 C. perfringens colony-forming units per gram of sampled material from C. perfringens-positive chickens ± SD. 3 Cecal samples from chickens housed in separate isolators and given saline but not the three-strain composite of C. perfringens. 4 Not determined.
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Intestinal lesions are found in chickens with necrotic enteritis. We did not examine the experimental chickens for lesions. Birds on the rye-based feed were observed to have decreased body weights compared to birds on the corn-based diet (data not shown), but increased mortality was not observed from the use of rye-supplemented feed. Previous controlled studies of chickens challenged with C. perfringens have also failed to induce mortality or other symptoms of necrotic enteritis, even though high C. perfringens numbers were reported in the intestinal tract (Cowen et al., 1987). In the field, necrotic enteritis is usually not noticed in a flock until mortality occurs in some birds at 2 wk of age. To sample the chickens after 3 wk postchallenge or to sample more birds would have increased the probability of detecting disease resulting from proliferation of the experimental C. perfringens strains. Clostridium perfringens is also associated with fibrosing hepatitis in broiler chickens, which results in liver abnormalities and condemnation of processed birds. C. perfringens has been isolated from the bile ducts and livers of affected birds (Hutchison and Riddel, 1990; Onderka et al., 1990). Because the study was restricted to chickens 3 wk of age or less, the chances of detecting liver abnormalities were low. In this study, no morphological differences were noted when comparing livers of birds that did or did not contain C. perfringens (data not shown). The frequency of isolation and recovery of this organism in the livers of birds on either of the two diets were low.
ACKNOWLEDGMENTS The author thanks Stephan Thayer, Poultry Disease Research Center, University of Georgia for supplying the C. perfringens strains and reviewing the manuscript and Douglas Cosby for his technical support.
REFERENCES Cowen, B. S., L. D. Schwartz, R. A. Wilson, and S. I. Ambrus, 1987. Experimentally induced necrotic enteritis in chickens. Avian Dis. 31:904–906. Fossum, O., K. Sandstedt, and B. E. Engstrom, 1988. Gizzard erosions as a cause of mortality in white leghorn chickens. Avian Pathol. 17:519–525. Gerdon, D., 1973. Effects of a mixed clostridial bacterin in incidence of gangrenous dermatitis. Avian Dis. 17:205–206. Harmon, S. M. 1984. Clostridium perfringens: Enumeration and identification. Ch.17. Pages 1701–1710 in: FDA Bacteriological Analytical Manual. Association of Official Analytical Chemists, Arlington, VA. Hofacre, C. L., J. D. French, R. K. Page, and O. J. Fletcher, 1986. Subcutaneous clostridial infection in broilers. Avian Dis. 30:620–622. Hutchison, T.W.S., and C. Riddel, 1990. A study of hepatic lesions in broiler chickens at processing plants in Saskatchewan. Can. Vet. J. 31:20–25. Kaldhusdal, M., and M. Hofshagen, 1992. Barley inclusion and avoparcin supplementation in broiler diets. 2. Clinical, pathological, and bacteriological findings in a mild form of necrotic enteritis. Poultry Sci. 71:1145–1153. Long, J. R., J. R. Pettit, and D. A. Barnum, 1974. Necrotic enteritis in broiler chickens II. Pathology and proposed pathogenesis. Can. J. Comp. Med. 38:467–475.
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sampling location (P ≤ 0.05) and between sampling day, intestinal sampling location, and diet (P ≤ 0.05). The low isolation rates of bacitracin-resistant C. perfringens throughout the intestinal tract and the low numbers in positive birds at 12, 24, and 36 h after the C. perfringens gavage indicate that the experimental strains given to the birds on a corn-based or 50% rye diet were for the most part quickly cleared by cell death, intestinal shedding, or some other mechanism. From 2 to 21 d after challenge, C. perfringens were found in the intestinal tract of only a few of the corn-fed birds. During this time period, the locus of most frequent isolation in C. perfringens-positive birds shifted from the upper to the lower parts of the intestinal tract; however, no consistent increase in total C. perfringens numbers over time was observed in any part of the intestinal tract of chickens given a corn-based diet. Also, numbers in C. perfringens-positive birds were low in all intestinal sampling locations. Clostridium perfringens numbers in positive birds were low in all portions of the intestinal tract of rye-fed birds at 12, 24, and 36 h after challenge of the birds with the C. perfringens composite culture but were higher in most parts of the intestinal tract after 48 h. C. perfringens proliferation occurred between 2 and 21 d in birds given the rye-supplemented feed with the highest counts observed in the jejunum, ileum, and ceca. Compared to birds on the corn-based diet, more birds on the rye-supplemented diet were contaminated with C. perfringens, contamination occurred earlier, C. perfringens was more widespread in the intestinal tract, and the mean counts were higher in these birds. C. perfringens numbers did not increase from 2 to 21 d in the crop, gizzard, or duodenum of birds on rye-supplemented feed, but counts in the jejunum, ileum, and ceca increased gradually by 3 to 5 log factors during this period. At 21 d, numbers of C. perfringens in the intestine of the experimental birds on rye feed were high, approaching those reported in chickens with necrotic enteritis (Kaldhusdal and Hofshagen, 1992). High numbers of C. perfringens in the feces of contaminated chickens would likely contribute to increased contamination of the skin and feathers of these birds, increased spread to adjacent animals, and increased spread of necrotic enteritis in a flock. The numbers and frequency of isolation of C. perfringens in the fresh feces of these birds were similar to those in the ileum, suggesting that fecal enumeration can be a predictor of intestinal numbers, especially when intestinal populations are high. Although routine enumeration of C. perfringens in fecal droppings would not be practical, enumeration could be used to indicate a potential problem in flocks that may be predisposed to necrotic enteritis or housed in a location with a history of problems known to be associated with C. perfringens. When flocks are detected with a high frequency of intestinal contamination and high intestinal numbers, therapeutic treatments administered before signs of disease and mortality may reduce some of the adverse effects of this organism, i.e., weight loss and mortality.
CLOSTRIDIUM PERFRINGENS INTESTINAL COLONIZATION Misir, R., and R. R. Marquardt, 1984. Factors affecting rye (Secale cereale L.) utilization in growing chicks. I. The influence of rye level, ergot and penicillin supplementation. Can. J. Anim. Sci. 58:691–701 Onderka, D. K., C. C. Langevin, and J. A. Hanson, 1990. Fibrosing cholehepatitis in broiler chickens induced by bile duct ligations or inoculation of Clostridium perfringens. Can. J. Vet. Res. 54:285–290. Parrish, W. E., 1961. Necrotic enteritis in the fowl (Gallus gallus domesticus). I. Histopathology of the disease and isolation of a strain of Clostridium welchii. J. Comp. Pathol. 71:377–393. Paulus, C., and J. P. Ruckebusch, 1996. Necrotic entertis (NE). Zootecn. Int. XIX(6):40–42. Riddell, C., and X.-M. Kong, 1992. The influence of diet on necrotic enteritis in broiler chickens. Avian Dis. 36:499–503. Shane, S. M., J. E. Gyimah, K. S. Harrington, and T. G. Snider, III, 1985. Etiology and pathogenesis of necrotic enteritis. Vet. Res. Commun. 9:269–287.
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Songer, J. G., 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 9:216–234. Stutz, M. W., and G. C. Lawton, 1984. Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks. Poultry Sci. 63:2036–2042. Takeda, T., T. Fukata, T. Miyamoto, K. Sasai, E. Baba, and A. Arakawa, 1995. The effects of dietary lactose and rye on cecal colonization of Clostridium perfringens in chicks. Avian Dis. 39:375–381. Truscott, R. B., and F. Al-Sheikhly, 1977. Reproduction and treatment of necrotic enteritis in broilers. Am J. Vet. Res. 38:857–861. Tsai, S. S., and M. C. Tung, 1981. An outbreak of necrotic enteritis in broiler chickens. J. Chinese Soc. Vet. Sci. 7:13–17. Tschirdewahn, B., S. Notermans, K. Wernars, and F. Untermann, 1991. The presence of enterotoxigenic Clostridium perfringens strains in feces of various animals. Internat. J. Food Microbiol. 14:175–178.
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(Key words: Clostridium perfringens, broiler chicken, intestine, rye) 2000 Poultry Science 79:843–849
poultry flocks worldwide that suffer from necrotic enteritis (Parrish, 1961; Long et al., 1974; Tsai and Tung, 1981). A better understanding of conditions that favor the proliferation of C. perfringens is important in determining the cause and spread of necrotic enteritis (Paulus and Ruckebusch, 1996). Factors that predispose flocks to necrotic enteritis include management stress, changes in dietary formulation, alteration of feeding programs, and subclinical intestinal coccidiosis (Shane et al., 1985). Grains such as wheat, barley, or rye, when added to a corn-based feed have been shown to inhibit weight gain, increase C. perfringens numbers, induce necrotic enteritis, and increase bird mortality (Misir and Marquardt, 1984; Riddell and Kong, 1992; Takeda et al., 1995). Little information is available about the change in C. perfringens numbers in birds before overt symptoms of the disease are observed, the relative numbers recovered from certain parts of the intestinal tract, or the roles of diet and fecal shedding in the spread of the disease. The current study was conducted to determine the rate and extent of increase in the intestinal population of C. perfringens, the numbers shed in fecal droppings, and the relationship of fecal numbers to numbers found in the
INTRODUCTION Clostridium perfringens is recognized as an enteric pathogen in humans, domestic animals, and wildlife worldwide (Songer, 1996). Necrotic enteritis, a disease of poultry caused by this organism, leads to the development of necrotic lesions in the gut wall, as well as increased mortality. Clostridium perfringens is also associated with other poultry diseases, including avian malignant disease, also known as gas edema disease (Hofacre et al., 1986), gizzard erosions (Fossum et al., 1988), and gangrenous dermatitis (Gerdon, 1973). It has been implicated in the growth depression of poultry (Stutz and Lawton, 1984) and the condemnation at slaughter due to liver abnormalities (Hutchison and Riddel, 1990; Onderka et al., 1990). A high number of C. perfringens in the intestinal tract and associated necrotic lesions have been detected in
Received for publication June 30, 1999. Accepted for publication February 23, 2000. 1 To whom correspondenceshould be addressed: scraven@asrr. arsusda.gov.
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ABSTRACT Commercial broiler chicks were given a three-strain composite of bacitracin-resistant Clostridium perfringens by oral gavage and were sampled periodically to determine the dynamics of C. perfringens colonization of the intestinal tract of broiler chickens and fecal shedding. After gavage, the chicks were divided into two groups and placed in isolators, one group received a traditional corn-based diet, and the other group received the same diet supplemented with 50% rye to place the birds under dietary stress. The numbers of bacitracin-resistant C. perfringens in various parts of the intestinal tract, liver, and feces were determined using a selective plating medium containing bacitracin. In chickens on the corn-based or 50% rye diet, C. perfringens was isolated infrequently from the various parts of the intestinal tract, liver, or fecal droppings during the first 36 h following the last gavage. For the 24 chickens
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intestinal tract during the first 3 wk of grow out. Experimental birds were given either a corn-based diet to simulate standard commercial conditions or a diet supplemented with 50% rye to expose the birds to dietary stress.
MATERIALS AND METHODS Experimental Design
Chickens Broiler chicks were housed in sanitized and temperature-controlled isolator units with wire mesh floors and positive air pressure for the duration of the experiment. Water was provided ad libitum through nipple waterers.
Feed Two types of feed were used: 1) a corn-based, unmedicated mash feed that contained 57.3% ground corn, 33.5% dehulled soybean meal, 3.2% poultry fat, and 3.0% poultry by-product meal with the remainder consisting of defluorinated phosphate, limestone, vitamin mix, salt, D,L-methionine, and trace minerals2 and 2) the cornbased feed supplemented with 50% (wt/wt) organic rye flakes.3 No attempts were made to equalize nutritional or caloric values for the two feed types. Feed was provided ad libitum for consumption throughout the trials.
2 Poultry Science Department, University of Georgia, Athens, GA 30602. 3 Arrowhead Mills, Hereford, TX 79045. 4 Difco Labs, Detroit, MI 48232. 5 Tekmar Co., Cincinnati, OH 45249. 6 Unipath, Co., Oxoid Division, Ogdensburg, NY 13669.
Clostridium perfringens 5227, 5229, and 5230, bacitracinresistant strains previously isolated from the outbreaks of necrotic enteritis, were obtained from S. Thayer, Poultry Disease Research Center, the University of Georgia, Athens, Georgia. The strains were inoculated from stock cultures into cooked meat medium4 and incubated at 37 C for 24 h. Equal volumes from the liquid portions of each of the three cultures were combined. From the composite culture, 0.1 mL (approximately 108 cells) was given to each chick in the C. perfringens-treated group by oral gavage on the day of hatch. A second dose of 0.1 mL of a fresh composite culture was given on the following day to each of these chicks.
Bacteriological Sampling At given intervals after the oral gavage with C. perfringens or saline (12, 24, and 36 h in Trial 1 and 2, 7, 14, and 21 d in Trials 1 and 2), birds were euthanatized by cervical dislocation to sample their intestinal tract. A 5 cm length of each intestinal sampling location was excised with a pair of surgical scissors, placed into a stomacher bag with sterile buffered peptone-water to give an initial 1/5 (wt/vol) dilution, and cut to allow homogenization of contents. The locations in the intestinal tract that were sampled included the crop, proventriculus, gizzard, duodenum, ileum, jejunum, and cecum. The intestinal segments and their contents were homogenized using the Stomacher Lab-blender 80.5 Serial dilutions were made in sterile saline. The liver and fresh fecal droppings were sampled in a similar manner. After stomaching, dilutions were prepared, and 0.1 mL of each dilution was placed into petri dishes and mixed with the selective plating medium consisting of Perfringens Agar Base6 with added egg yolk suspension, 20 µg/mL kanamycin, and 20 µg/mL bacitracin. Agar plates were overlaid with the same plating medium but without egg yolk. After anaerobic incubation for 48 h at 37 C, black colonies with white halos, which were indicative of lecithinase activity, were counted as C. perfringens. In a preliminary study conducted to determine the effectiveness of the recovery medium for enumerating bacitracin-resistant C. perfringens in intestinal contents, cecal contents from unchallenged 3-wk-old chickens were inoculated with the C. perfringens composite, diluted, and plated on the recovery medium containing egg yolk, kanamycin, and bacitracin. An uninoculated portion was plated as a control. Lecithinase-positive and -negative colonies were picked and confirmed according to Harmon (1984). Isolates that were lecithinase positive, displayed motility, reduced nitrate to nitrite, produced acid and gas from lactose, and liquified gelatin within 48 h were considered C. perfringens. Fifty colonies of sulfite-reducing bacteria (black) demonstrating a lecithinase reaction (white halo) and fifty colonies of sulfite-reducing bacteria with no lecithinase reaction were characterized (data not shown). None of the black, lecithinase-negative colonies
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In each of two trials, 120 day-of-hatch chicks obtained from a local broiler chicken hatchery were divided into four equal groups, and each group was placed in a separate isolator. Chicks in two isolators were maintained on corn-based feed, and chicks in the other two isolators were on 50% (wt/wt) rye feed. For each feed treatment, chicks in one of the two isolators were given C. perfringens, and the chicks in the other isolator were given a saline control. In Trial 1, chickens were euthanatized at 12, 24, or 36 h or at 2, 7, 14, or 21 d after C. perfringens administration and intestinal samples were prepared to determine counts of bacitracin-resistant C. perfringens with a selective plating medium. In Trial 2, chickens were not sampled at 12, 24, or 36 h but were sampled as in Trial 1 at 2, 7, 14, and 21 d. At 12 h in Trial 1 and 21 d in Trials 1 and 2, control chicks receiving no C. perfringens were also sampled and plated in the same manner. Three birds per sampling time per diet per trial were sampled for a total of 66 birds. More were not used because previous studies have shown that the numbers of C. perfringens were higher in chickens on a diet containing rye compared to numbers in chickens on a corn-based diet.
Clostridium perfringens Strains
CLOSTRIDIUM PERFRINGENS INTESTINAL COLONIZATION
were confirmed as C. perfringens. Of those colonies, 65% were facultative anaerobes, and 35% were strict anaerobes. All of the black, lecithinase-positive colonies were determined to be strict anaerobes. Of those, 86% were confirmed as C. perfringens. Some of the remaining 14% were likely false negatives because black, lecithinase-positive colonies did not appear on plates of similar dilutions of a cecal sample not inoculated with C. perfringens (data not shown). When a culture of the three-strain composite of C. perfringens without cecal contents was plated on the recovery agar containing kanamycin and bacitracin, numbers were similar to those on basal medium without antibiotics (data not shown). The results indicated that the recovery medium was selective for and not inhibitory to the experimental strains of C. perfringens.
Statistical Analysis
RESULTS At 12, 24, and 36 h after the chickens were gavaged with the experimental three-strain composite of C. perfringens, few C. perfringens were recovered from the intestinal tracts of birds fed the corn-based feed (Table 1). Only the crop and ileum of one bird tested positive for C. perfringens. C. perfringens was recovered occasionally during the first 36 h from chickens on 50% rye feed and in more birds and in more intestinal sampling locations than for birds on corn-based feed (Table 1). The numbers of C. perfringens from any portion of the intestinal tract at 12 to 36 h were ≤log10 5.5/g of intestinal sample from birds on either of the two diets. The frequency of C. perfringens recovered from most parts of the intestinal tract decreased between 12 and 36 h regardless of the diet. C. perfringens was not recovered from the ceca of control birds. For the four sampling periods of 2, 7, 14, and 21 d postchallenge, C. perfringens was recovered on at least two of the four sampling periods from each portion of the intestinal tract of some of the birds challenged with the three-strain composite and subsisting on corn-based feed (Table 2). None were detected in control ceca. The
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Dataxiom Software Inc., Los Angeles, CA 90010.
highest frequency of isolation was found at Day 7 in the crop (six of six birds) and ileum (five of six birds). On Day 7 C. perfringens was found less frequently in the proventriculus, gizzard, duodenum, jejunum, and ceca and on Days 2, 14, and 21 for the crop and ileum. In those intestinal sampling locations where two to six birds were positive for C. perfringens, mean C. perfringens numbers for those birds on a given day from 2 to 21 d were similar in the different locations of the intestinal tract sampled. An exception was Day 14 on which fecal counts were significantly higher than ileal counts. No significant increases in mean C. perfringens numbers were observed in C. perfringens-positive birds on a corn-based diet during the period of 2 to 21 d. No consistent increase in isolation rates were observed, only a transient increase in recovery of C. perfringens from the crop, ileum, and feces on Day 7. In the rye-fed birds, all intestinal sampling locations of one or more birds contained C. perfringens on Day 2 (Table 3). By Day 7, C. perfringens could be found in the crop and ileum of all six chickens sampled and in all intestinal sampling locations of two of the six birds. Results were similar to those observed for corn-fed birds at Day 7 (Table 2), in that the highest frequency of recovery of C. perfringens occurred in the crop and the ileum. From 7 to 21 d, the frequency of isolation from the crop decreased for birds on the rye-supplemented diet, but the frequency of isolation from other intestinal sampling sites remained the same or increased. In contrast to the C. perfringens counts from positive birds on corn-based feed, the mean numbers of C. perfringens in positive birds on rye-supplemented feed varied in different parts of the intestinal tract on Days 7, 14, and 21. Mean numbers in positive birds increased from 2 to 21 d in the ileum and ceca by as much as 3.3 to 4.9 log factors with mean counts in birds at 21 d ranging from log10 6.8 to 7.9/g in these intestinal locations. The numbers of C. perfringens detected in the fecal droppings of birds generally reflected the numbers found in the ileum. In chickens on the corn-based and 50% rye diets, C. perfringens was not detected in the feces at 12 to 36 h (Table 1). On 2, 7, 14, and 21 d, the frequency of isolation from feces of chickens on a corn-based diet was 0 to 3 out of 6 sampled birds with numbers ≤ log10 6.2/ g for positive samples. The frequency of isolation from samples of the ileum was one to five of six birds with numbers ≤ log10 4.3/g (Table 2). The frequency of isolation from the feces of birds on the 50% rye diet was four to five out of six sampled birds with numbers ranging from log10 3.8 to 7.1/g for C. perfringens-positive samples compared to an isolation frequency of two to six out of six in the ileum with mean numbers ranging from log10 3.7 to 7.5 in positive samples (Table 3). In this study, the frequency of isolation of C. perfringens from the liver of chickens on either diet was low (Tables 1 to 4). The numbers found in C. perfringens-positive livers were also low. A comparison of the occurrence of C. perfringens in chickens on a corn-based diet (Tables 1 and 2) and on a 50% rye diet (Tables 1 and 3) indicated that mean numbers in positive birds were higher throughout most of the
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Statistical analyses were performed using Statistica for Windows software.7 Student’s t-test was used to determine significant differences for C. perfringens numbers between different intestinal sampling locations and different sampling days. Significant differences in the frequency of recovery of C. perfringens from intestinal samples of chickens were determined by pooling the results from Days 2, 7, 14, and 21 for each intestinal sampling location of birds on a given diet. Fisher’s exact test was applied to the pooled data that had been organized into 2 × 2 contingency tables. Significance for both tests was determined at P ≤ 0.05.
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CRAVEN TABLE 1. Occurrence of Clostridium perfringens in chickens at 12, 24, and 36 h after final gavage and when fed a corn-based or 50% rye diet in Trial 1 Chickens1 positive for C, perfringens Intestinal sampling location
Corn-based diet 12 h
Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Ceca control3 Feces Liver
2
1 (5.5) 0 0 0 0 1 (2.7) 0 0 0 0
50% rye diet
24 h
36 h
12 h
0 0 0 0 0 0 0 ND4 0 0
0 0 0 0 0 0 0 ND 0 0
2 0 2 0 1 1 1 0 0 1
(3.3 ± 0.8) (2.9) (2.3) (2.5) (5.5) (3.3)
24 h
36 h
0 0 0 0 1 (2.9) 0 1 (4.6) ND 0 0
0 1 (5.3) 0 1 (4.8) 0 0 0 ND 0 1 (4.2)
1
Out of three chickens per diet per day. Values in parentheses = mean log10 C. perfringens colony-forming units per gram of sample material from C. perfringens-positive chickens ± SD. 3 Cecal samples from chickens housed in separate isolators given saline but not the three-strain composite of C. perfringens. 4 Not determined. 2
numbers at 2 d, the high numbers at 21 d, and the numbers from Days 7 and 14 for the rye-fed birds.
DISCUSSION Addition of rye to the diet of chickens has been reported to increase numbers of C. perfringens in the cecal contents (Takeda et al., 1995). Our current study confirms and extends these findings by demonstrating an increase in numbers of C. perfringens from 2 to 21 d of age in the ileum, ceca, and feces of birds given a diet containing 50% rye. Also, a higher frequency of recovery through most of the intestinal tract and higher numbers in the jejunum, ileum, and ceca of C. perfringens-positive birds occurred as compared to corresponding numbers in birds on a corn-based diet.
TABLE 2. Occurrence of Clostridium perfringens in chickens at 2, 7, 14, and 21 d after final gavage and when fed a corn-based diet in Trials 1 and 2 Intestinal sampling location Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Ceca control3 Feces Liver
Chickens1 positive for C. perfringens 2d
7d
0 2 (4.5 ± 2 (3.4 ± 2 (6.0 ± 0 1 (2.8) 1 (3.4) ND4 0 2 (3.9 ±
6 (3.9 ± 1.1) 0 1 (3.5) 0 1 (2.3) 5 (2.9 ± 0.7)a 1 (3.9) ND 3 (4.9 ± 1.7)a 0
a,2
0.8)a 1.9)a 1.0)a
1.1)a
14 d
21 d
1 (4.0) 1 (2.7) 1 (2.0) 1 (3.7) 1 (3.5) 2 (3.0)b 0 ND 2 (4.5 ± 0.2)a 0
0 0 2 1 2 3 2 0 1 0
(3.1 ± (2.0) (3.0 ± (4.3 ± (4.9 ±
0.3)a 1.4)a 1.2)a 2.7)a
(6.2)
a Means of intestinal and fecal samples within columns with no common lowercase superscripts differ significantly (P ≤ 0.05). 1 Out of six chickens, three chickens per trial per day. 2 Values in parentheses = mean log10 C. perfringens colony-forming units per gram of sampled material from C. perfringens-positive chickens ± SD. 3 Cecal samples from chickens housed in separate isolators and given saline but not the three-strain composite of C. perfringens. 4 Not determined.
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intestinal tract for chickens on the rye-supplemented diet and suggested that recovery rates were also higher. A direct comparison of birds on the two diets with data pooled from Days 2, 7, 14, and 21 is shown in Table 4. Of the 24 birds on each of the diets sampled, the recovery of C. perfringens was significantly higher in the crop, proventriculus, duodenum, jejunum, ileum, ceca, and feces of birds on the 50% rye diet. Recovery rates from the gizzard and liver were not significantly different in birds on the two diets. The overall numbers (mean log10 cfu/ g of intestinal material from all positive birds) were significantly higher (by 2.6 log factors) in the jejunum of birds given 50% rye. The overall numbers in the ileum and ceca were 2.7 to 2.8 log factors higher in birds on the rye diet, but this difference was not significant due to the large variation that resulted by averaging the low
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CLOSTRIDIUM PERFRINGENS INTESTINAL COLONIZATION TABLE 3. Occurrence of Clostridium perfringens in chickens at 2, 7, 14, and 21 d after final gavage and when fed a 50% rye diet in Trials 1 and 2 Intestinal sampling location Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Ceca control3 Feces Liver
Chickens1 positive for C. perfringens 2d
7d 2
1 (4.8) 4 (4.3 ± 0.7)ab,x 2 (4.6 ± 0.6)a,x 4 (4.4 ± 1.5)ab,x 3 (3.9 ± 1.6)ab,x 2 (3.7)b,y 1 (3.0) ND4 4 (3.8 ± 0.9)ab,y 0
14 d
6 (3.5 ± 2 (4.5 ± 2 (3.4 ± 4 (5.1 ± 5 (4.6 ± 6 (4.9 ± 3 (6.9 ± ND 5 (5.0 ± 2 (3.2)
bcx,2
1.8) 2.7)abc,x 0.5)c,y 1.3)abc,x 1.7)abc,x 2.2)abc,xy 0.5)a,y 0.5)b,x
5 (4.2 4 (4.0 3 (5.0 4 (5.2 6 (5.7 5 (6.7 5 (6.5 ND 5 (6.2 0
21 d ± ± ± ± ± ± ±
c,x
1.5) 1.4)c,x 1.7)abc,xy 2.0)abc,x 1.0)abc,xy 0.8)a,x 0.5)ab,y
± 1.5)abc,xy
2 3 5 5 5 6 6 0 5 2
(4.8 (4.5 (4.0 (5.0 (6.8 (7.5 (7.9
± ± ± ± ± ± ±
0.3)c,x 2.4)bc,x 1.7)c,xy 2.2)abc,x 0.7)ab,x 1.0)ab,x 0.7)a,x
(7.1 ± 1.8)ab,x (3.4 ± 0.9)
a,b
Clostridium perfringens is commonly found in the intestinal tract of poultry (Tschirdewahn et al., 1991), but the occurrence of the poultry disease, necrotic enteritis, is sporadic (Cowen et al., 1987). A complete understanding of the factors contributing to infection of the intestinal tract and subsequent events leading to necrosis is lacking (Truscott and Al-Sheikhly, 1977). Examination of the intestinal tract of affected birds in flocks with this disease and in controlled studies shows that high numbers of C. perfringens cells are present and are intimately associated with damaged tissue. Because these high numbers are not generally found in the intestinal tract of birds from healthy flocks, conditions favoring proliferation of C. perfringens in the intestinal tract appear to be essential for disease outbreaks. In evaluating increases in intestinal numbers of C. perfringens and effects of independent variables in this study,
numbers in C. perfringens-positive birds only were considered because the value for negative birds would skew the means, increase the variability among means, and perhaps mask changes or differences in C. perfringens numbers in that portion of the bird population that was contaminated by this organism. An analysis of C. perfringens numbers in positive birds by Student’s t-test and frequency of recovery of C. perfringens by Fisher’s exact test indicated that the intestinal sampling location, diet, and day of sampling could influence the occurrence of C. perfringens (P ≤ 0.05). In contrast, when an ANOVA of the total bird population (C. perfringens-positive and negative birds) was conducted, the intestinal sampling location (but not the sampling day, diet, or trial number) was shown to have a significant effect on C. perfringens numbers (P ≤ 0.05) (data not shown). However, there were interactions between sampling day and intestinal
TABLE 4. Occurrence of Closridium perfringens in chickens from Trials 1 and 2 pooled for sampling days 2, 7, 14, and 21 d. Chickens were maintained ad libitum on a corn-based or 50% rye diet Intestinal sampling location Crop Proventriculus Gizzard Duodenum Jejunum Ileum Ceca Feces Liver
Recovery of C. perfringens1 Corn-based diet 7 3 6 4 4 11 4 6 2
50% rye diet a
14 13a 12 17a 19a 19a 15a 19a 4
Mean cfu/g of intestinal material2 Corn-based diet 3.9 3.9 3.1 4.2 3.0 3.3 4.2 5.0 3.9
± ± ± ± ± ± ± ± ±
1.0 1.2 1.0 1.8 1.0 0.9 1.5 1.2 1.1
50% rye diet 4.0 4.3 4.4 4.9 5.6 6.1 6.9 6.1 3.0
± ± ± ± ± ± ± ± ±
1.2 1.5 1.5 1.7 1.4a 2.0 1.5 1.5 0.9
a Superscripts indicate a significant difference between chickens on the different diets for the recovery of C. perfringens or mean colony-forming units of C. perfringens per gram (P ≤ 0.05). 1 Number of chickens positive for C. perfringens at a given intestinal location of the 24 total chickens on a given diet and sampled from 2 to 21 d. 2 Mean log10 C. perfringens colony-forming units per gram of material sampled at a given intestinal location for the positive birds of the 24 chickens on a given diet and sampled from 2 to 21 d.
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Means of intestinal and fecal samples within columns with no common superscript differ significantly (P ≤ 0.05). x,y Means within rows with no common superscript differ significantly (P ≤ 0.05). 1 Out of six chickens, three chickens per trial per day. 2 Values in parentheses = mean log10 C. perfringens colony-forming units per gram of sampled material from C. perfringens-positive chickens ± SD. 3 Cecal samples from chickens housed in separate isolators and given saline but not the three-strain composite of C. perfringens. 4 Not determined.
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Intestinal lesions are found in chickens with necrotic enteritis. We did not examine the experimental chickens for lesions. Birds on the rye-based feed were observed to have decreased body weights compared to birds on the corn-based diet (data not shown), but increased mortality was not observed from the use of rye-supplemented feed. Previous controlled studies of chickens challenged with C. perfringens have also failed to induce mortality or other symptoms of necrotic enteritis, even though high C. perfringens numbers were reported in the intestinal tract (Cowen et al., 1987). In the field, necrotic enteritis is usually not noticed in a flock until mortality occurs in some birds at 2 wk of age. To sample the chickens after 3 wk postchallenge or to sample more birds would have increased the probability of detecting disease resulting from proliferation of the experimental C. perfringens strains. Clostridium perfringens is also associated with fibrosing hepatitis in broiler chickens, which results in liver abnormalities and condemnation of processed birds. C. perfringens has been isolated from the bile ducts and livers of affected birds (Hutchison and Riddel, 1990; Onderka et al., 1990). Because the study was restricted to chickens 3 wk of age or less, the chances of detecting liver abnormalities were low. In this study, no morphological differences were noted when comparing livers of birds that did or did not contain C. perfringens (data not shown). The frequency of isolation and recovery of this organism in the livers of birds on either of the two diets were low.
ACKNOWLEDGMENTS The author thanks Stephan Thayer, Poultry Disease Research Center, University of Georgia for supplying the C. perfringens strains and reviewing the manuscript and Douglas Cosby for his technical support.
REFERENCES Cowen, B. S., L. D. Schwartz, R. A. Wilson, and S. I. Ambrus, 1987. Experimentally induced necrotic enteritis in chickens. Avian Dis. 31:904–906. Fossum, O., K. Sandstedt, and B. E. Engstrom, 1988. Gizzard erosions as a cause of mortality in white leghorn chickens. Avian Pathol. 17:519–525. Gerdon, D., 1973. Effects of a mixed clostridial bacterin in incidence of gangrenous dermatitis. Avian Dis. 17:205–206. Harmon, S. M. 1984. Clostridium perfringens: Enumeration and identification. Ch.17. Pages 1701–1710 in: FDA Bacteriological Analytical Manual. Association of Official Analytical Chemists, Arlington, VA. Hofacre, C. L., J. D. French, R. K. Page, and O. J. Fletcher, 1986. Subcutaneous clostridial infection in broilers. Avian Dis. 30:620–622. Hutchison, T.W.S., and C. Riddel, 1990. A study of hepatic lesions in broiler chickens at processing plants in Saskatchewan. Can. Vet. J. 31:20–25. Kaldhusdal, M., and M. Hofshagen, 1992. Barley inclusion and avoparcin supplementation in broiler diets. 2. Clinical, pathological, and bacteriological findings in a mild form of necrotic enteritis. Poultry Sci. 71:1145–1153. Long, J. R., J. R. Pettit, and D. A. Barnum, 1974. Necrotic enteritis in broiler chickens II. Pathology and proposed pathogenesis. Can. J. Comp. Med. 38:467–475.
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sampling location (P ≤ 0.05) and between sampling day, intestinal sampling location, and diet (P ≤ 0.05). The low isolation rates of bacitracin-resistant C. perfringens throughout the intestinal tract and the low numbers in positive birds at 12, 24, and 36 h after the C. perfringens gavage indicate that the experimental strains given to the birds on a corn-based or 50% rye diet were for the most part quickly cleared by cell death, intestinal shedding, or some other mechanism. From 2 to 21 d after challenge, C. perfringens were found in the intestinal tract of only a few of the corn-fed birds. During this time period, the locus of most frequent isolation in C. perfringens-positive birds shifted from the upper to the lower parts of the intestinal tract; however, no consistent increase in total C. perfringens numbers over time was observed in any part of the intestinal tract of chickens given a corn-based diet. Also, numbers in C. perfringens-positive birds were low in all intestinal sampling locations. Clostridium perfringens numbers in positive birds were low in all portions of the intestinal tract of rye-fed birds at 12, 24, and 36 h after challenge of the birds with the C. perfringens composite culture but were higher in most parts of the intestinal tract after 48 h. C. perfringens proliferation occurred between 2 and 21 d in birds given the rye-supplemented feed with the highest counts observed in the jejunum, ileum, and ceca. Compared to birds on the corn-based diet, more birds on the rye-supplemented diet were contaminated with C. perfringens, contamination occurred earlier, C. perfringens was more widespread in the intestinal tract, and the mean counts were higher in these birds. C. perfringens numbers did not increase from 2 to 21 d in the crop, gizzard, or duodenum of birds on rye-supplemented feed, but counts in the jejunum, ileum, and ceca increased gradually by 3 to 5 log factors during this period. At 21 d, numbers of C. perfringens in the intestine of the experimental birds on rye feed were high, approaching those reported in chickens with necrotic enteritis (Kaldhusdal and Hofshagen, 1992). High numbers of C. perfringens in the feces of contaminated chickens would likely contribute to increased contamination of the skin and feathers of these birds, increased spread to adjacent animals, and increased spread of necrotic enteritis in a flock. The numbers and frequency of isolation of C. perfringens in the fresh feces of these birds were similar to those in the ileum, suggesting that fecal enumeration can be a predictor of intestinal numbers, especially when intestinal populations are high. Although routine enumeration of C. perfringens in fecal droppings would not be practical, enumeration could be used to indicate a potential problem in flocks that may be predisposed to necrotic enteritis or housed in a location with a history of problems known to be associated with C. perfringens. When flocks are detected with a high frequency of intestinal contamination and high intestinal numbers, therapeutic treatments administered before signs of disease and mortality may reduce some of the adverse effects of this organism, i.e., weight loss and mortality.
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Songer, J. G., 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 9:216–234. Stutz, M. W., and G. C. Lawton, 1984. Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks. Poultry Sci. 63:2036–2042. Takeda, T., T. Fukata, T. Miyamoto, K. Sasai, E. Baba, and A. Arakawa, 1995. The effects of dietary lactose and rye on cecal colonization of Clostridium perfringens in chicks. Avian Dis. 39:375–381. Truscott, R. B., and F. Al-Sheikhly, 1977. Reproduction and treatment of necrotic enteritis in broilers. Am J. Vet. Res. 38:857–861. Tsai, S. S., and M. C. Tung, 1981. An outbreak of necrotic enteritis in broiler chickens. J. Chinese Soc. Vet. Sci. 7:13–17. Tschirdewahn, B., S. Notermans, K. Wernars, and F. Untermann, 1991. The presence of enterotoxigenic Clostridium perfringens strains in feces of various animals. Internat. J. Food Microbiol. 14:175–178.
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