Necrotic Enteritis In Broiler Chickens Research Articles

Clostridium perfringens Type A from Broiler Chicken with Necrotic Enteritis

This paper reports the investigation of necrotic enteritis (NE) in six broiler chickens of age two to three weeks old, died in Jowai poultry farm, Meghalaya, India. Initially, scanning electron microscopy (SEM) was performed to observe the morphological changes within the intestine. Intestinal contents and liver samples from dead chicken were investigated for isolation of bacteria and their virulence determinant. The SEM analysis of infected intestine revealed massive necrosis and complete destruction of the intestinal villi within the intestinal mucosa. Bacterial isolation confirmed the causative agent as C. perfringens in NE. All the isolates harboured single and double plasmid deoxyribonucleic acid with identical 45.2kb common plasmid. In polymerase chain reaction (PCR) assay all 10 clinical isolates harboured alpha toxin gene (cpa) of C. perfringens, however, four isolates also carried additional beta2 toxin gene (cpb2). None of the isolates were positive for beta, epsilon, iota and enterotoxin genes. PCR analysis revealed that all isolates derived from NE belonged to C. perfringens type A. The partial cpa gene sequence analysis showed 97.6 to 100% homology among the C. perfringens isolates. The study confirmed that C. perfringens type A is the most predominant one associated with necrotic enteritis in broiler chickens in Meghalaya, India and the alpha toxin (CPA) might play a significant role in the pathogenesis of the disease in broiler chicken.

Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue

The present study revealed several previously not recognized etiological details in the development of necrotic enteritis (NE) in broilers. We provide evidence that the pathological process leading to mucosal epithelium necrosis follows morphologically distinct phases commencing at the basal domain of the mucosal epithelium and then progressively invading the entire lamina propria. Initially mucosal epithelium appears normal, but as the pathological changes progress throughout the lamina propria, the adjacent enterocytes begin to show features of necrotic cell death and the necrotic process of the epithelium progresses from being focal to locally extensive. Ultra-structural examination showed that primary changes occur at the level of basal and lateral domains of the enterocytes, whereas the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that the mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of the destruction of lamina propria, the extra-cellular matrix, and intercellular junctions. The nature of these morphological changes indicates that initiation of the pathological process leading to NE involves proteolytic factors affecting the extra-cellular matrix and cellular junctions. Further studies revealed that, indeed, the elevated activity of collagenolytic enzymes in the mucosal milieu and in intestinal tissue represents an integral component of the pathological process leading to NE. In the first instance we discovered that Clostridium perfringens strains isolated from field cases of NE secrete several potent collagenolytic enzymes. In the second instance we observed that, in comparison to controls, broilers challenged with C. perfringens isolated from field cases of NE show high levels of several collagenolytic enzymes in the intestinal tissue. A major component of the overall collagenolytic activity detected in the intestinal tissue was identified by zymography as matrix metalloproteinases (MMPs). Dominant activity was associated with MMP-2. We confirmed using immuno-histochemistry that this enzyme is expressed at high levels in mucosal tissue showing signs of NE. The high levels of collagenolytic activities, in particular associated with MMP-2, demonstrated in our studies are consistent with the nature of morphological changes observed primarily in extra-cellular matrix (ECM) at the basal domain of enterocytes, as well lateral domains of enterocytes. The lack of changes at the level of apical domain of mucosal epithelium indicates that the lipolytic aspect of alpha toxin in NE is not an essential factor in primary lesions development. Taken together, our findings indicate that the early lesions leading to NE are associated with virulence factors that induce proteolytic activity, rather than lipolytic activity.

Quantification of Cell Proliferation and Alpha-Toxin Gene Expression ofClostridium perfringensin the Development of Necrotic Enteritis in Broiler Chickens

Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b(0) + b(1)X - b(2)X(2). The average C. perfringens count of 5 log(10) CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.

Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era

The use of in-feed antibiotics has until now been the main strategy for controlling Clostridium perfringens-associated necrotic enteritis in poultry. But approvals for the inclusion of non-therapeutic antibiotics in poultry feed are fast disappearing worldwide due to fear of development of antibiotic-resistant microbes. Public concern about the threat of antibiotic-resistant pathogens has forced the poultry industry to consider various other alternatives. Strategies to control necrotic enteritis in the absence of antibiotic growth promoters, without resorting to the use of prophylactic or therapeutic treatment, have centered upon dietary and management practices. Among the candidate replacements for antibiotics are competitive exclusion products, probiotics, prebiotics, organic acids, enzymes, plant extracts, hen egg antibodies, bacteriophages and vaccination. Chemical composition of the diet related to cereal grain selection, protein source and amino acid profile may influence disease propensity and consideration of diet chemical composition might be helpful in formulation of poultry diets that reduce the incidence of necrotic enteritis. To date, no single satisfactory non-antibiotic measure against C. perfringens has been identified. Combined with good hygiene management of poultry houses, consideration of diet composition and application of antibiotic alternatives might be effective to some extent in maintaining production and controlling necrotic enteritis. This paper will review some of the potential strategies that are available for controlling necrotic enteritis in broiler chickens without using antibiotic growth promoters.

Dietary glycine concentration affects intestinal Clostridium perfringens and lactobacilli populations in broiler chickens1

Previous studies have reported that intestinal populations of Clostridium perfringens, the causative agent of necrotic enteritis (NE), are correlated with diets high in glycine. To establish a direct causative link, 3 trials were conducted to examine the effect of dietary glycine levels on gut populations of C. perfringens, alpha-toxin production, and NE lesion scores in broiler chickens. In trials 1 and 2, 12 groups of 4 birds were fed 4 different ideal protein-balanced diets formulated to contain 0.75, 1.58, 3.04, or 4.21% glycine from d 14 to 28 of age. In trial 3, 24 groups of 4 birds were given 6 different ideal protein-balanced diets formulated to contain 0.50, 0.75, 1.00, 1.50, 2.00, or 4.00% glycine. All birds were orally challenged with a broth culture of C. perfringens type A on d 1 and between d 14 and 21 of age and killed on d 28. The majority of birds showed clinical signs of NE with 4.16 to 8.33% mortality in the 3 trials. The highest mortality and intestinal lesion scores were observed in chickens receiving 3.04% glycine in trials 1 and 2, and 4.00% glycine in trial 3. Clostridium perfringens populations in the cecum varied quadratically with increasing dietary glycine, with the maximal response seen at 3.30,3.89, and 3.51% dietary glycine in trials 1, 2, and 3, respectively. Numbers of lactobacilli in cecum declined significantly (P < 0.05) with increasing levels of glycine. The results suggest that dietary glycine level has a significant effect on C. perfringens and lactobacilli populations and may be a predisposing factor for NE in broiler chickens.

Live attenuated vaccine-based control of necrotic enteritis of broiler chickens

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.

The efficacy of bacitracin methylene disalicylate when fed in combination with narasin in the management of necrotic enteritis in broiler chickens

The efficacy of bacitracin methylene disalicylate (BMD) in the management of necrotic enteritis (NE) when fed in combination with narasin was investigated in a floor-pen study of 2,000 broiler chickens using a Clostridium perfringens inoculum challenge model. Treatments consisted of 1) nonchallenged-nonmedicated; 2) challenged-nonmedicated; 3) challenged-narasin (70 ppm); 4) challenged-BMD (55 ppm); 5) challenged-narasin (70 ppm) + BMD (55 ppm). Medication was provided in the feed from Day 0 to trial termination on Day 41. C. perfringens challenge occurred from Day 14 to 16. BMD and narasin, fed alone and in combination, reduced (P < 0.05) mortality due to NE when compared to challenged-nonmedicated birds. NE lesion scores (Days 0 through 41) were lower among birds fed BMD and narasin, alone and in combination, compared to challenged-nonmedicated birds. Improvements in NE mortality and NE lesion scores were greatest for the BMD + narasin-medicated birds, followed by the BMD-alone, and then narasin-alone treated birds. BMD and narasin, alone and in combination, provided improvements (P < 0.05) in average daily gains over the entire study (Days 0 to 41). The results of this study demonstrate the effectiveness of BMD and narasin in the management of NE in broiler chickens.

Efficacy of In-Feed Tylosin Phosphate for the Treatment of Necrotic Enteritis in Broiler Chickens

The efficacy of tylosin phosphate for the treatment of necrotic enteritis (NE) was investigated in a floor pen study of 2,000 broiler chickens. A model in which Clostridium perfringens was administered in the feed on Days 14 to 16 was used to initiate an outbreak of NE. Treatments, allocated at the pen level in a randomized complete block design, consisted of five levels of tylosin phosphate (0, 50, 100, 200, or 300 ppm) administered in the feed on Days 15 to 22, following the identification of an outbreak of NE on Day 15. Mortality due to NE was significantly reduced (P < 0.05) for medicated birds at all dose levels of tylosin phosphate compared to unmedicated birds. Mean NE lesion scores on Day 17 were significantly reduced (P < 0.05) by all levels of tylosin treatment compared to those of unmedicated birds, decreasing linearly from 2.66 at 0 ppm to 0.38 at 100 ppm and 0 at higher doses. Tylosin at all levels provided improvement in Day 29 body weight, average daily gain, feed to gain ratio, and average daily feed intake compared to unmedicated birds. The results of this study provide evidence that tylosin phosphate, when administered in feed, is effective in the treatment of clinical outbreaks of NE in broiler chickens and suggest that the optimal dose for this purpose is 100 ppm.

Efficacy of Narasin in the Prevention of Necrotic Enteritis in Broiler Chickens

The efficacy of narasin in the control of necrotic enteritis (NE) was investigated in a floor pen study of 2000 broiler chickens using a Clostridium perfringens feed inoculum challenge model. Treatments were 1) nonmedicated, nonchallenged; 2) nonmedicated, challenged; 3) narasin, nonchallenged; 4) narasin, challenged. Narasin was administered at 70 ppm in the feed from day 0 to trial termination on day 41. Challenge inoculum contained approximately 1 x 10(8) colony-forming units CP/ml and was administered from day 14 to day 16. In the unmedicated groups, challenged birds had significantly (P < 0.05) lower mean body weight and reduced feed efficiency at day 21 and significantly (P < 0.01) higher cumulative NE mortality at day 41 compared with unchallenged. Similarly, among unmedicated birds, those challenged had a significantly (P < 0.01) higher mean NE score on day 17 and significantly (P < 0.05) higher mean huddling scores on days 15-17 than unchallenged. Among challenged birds, those fed narasin had significantly (P < 0.05) higher mean body weight and improved feed efficiency at days 21 and 41 and significantly (P < 0.01) lower cumulative NE mortality at day 41 than unmedicated. Similarly, among challenged birds, those receiving narasin had a lower mean NE score on day 17 (P > 0.05) and significantly (P < 0.05) lower huddling scores on days 16 and 17 than unmedicated. Coccidiosis lesion scores were zero for birds euthanatized from all treatment groups on day 17, suggesting that the beneficial effects of narasin were not due to prevention of coccidiosis. This study thus provides evidence that narasin is effective in the prevention of necrotic enteritis in broiler chickens.

Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables.

The effect of Clostridium perfringens challenge, number of challenge days, and pre-challenge antibiotic treatment on the induction of necrotic enteritis in broiler chickens raised on litter was studied, and the relationship between bacterial counts and frequency of gut lesions was evaluated. Specific intestinal lesions in randomly selected birds were present despite a lack of disease-specific mortality. Challenge, number of challenge days and frequency of lesions were associated with median counts of C. perfringens. The effect of pre-challenge C. perfringens counts and antibiotics cannot be evaluated unless procedures for the control of pre-challenge infection and methods for the differentiation between wild-type and challenge strains are established.

Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables

The effect of Clostridium perfringens challenge, number of challenge days, and pre-challenge antibiotic treatment on the induction of necrotic enteritis in broiler chickens raised on litter was studied, and the relationship between bacterial counts and frequency of gut lesions was evaluated. Specific intestinal lesions in randomly selected birds were present despite a lack of disease-specific mortality. Challenge, number of challenge days and frequency of lesions were associated with median counts of C. perfringens. The effect of pre-challenge C. perfringens counts and antibiotics cannot be evaluated unless procedures for the control of pre-challenge infection and methods for the differentiation between wild-type and challenge strains are established.

Association between cereal contents in the diet and incidence of necrotic enteritis in broiler chickens in Norway

The quarterly cumulative incidence of necrotic enteritis in the broiler chicken population of south-eastern Norway varied between zero and 34.8% during the years 1969–1989. Two epidemics appeared during the study period. The first and second epidemics lasted approximately 5 and 2 years and had peak incidences of 34.8% and 25.3%, respectively. The median incidence of all quarters was 1.2%, reflecting the generally low incidence during non-epidemic quarters. Univariable regression analysis indicated that necrotic enteritis occurred more often during the months October–March than during the months April–September. The inclusion levels of maize, barley, oats and wheat in broiler starter feed varied between zero and 38.0%, zero and 20.0%, zero and 26.0%, and 6.0% and 38.6%, respectively. A single primary feed variable was specified, based on a ratio comprising weighted values of maize, barley and wheat. This variable showed a striking covariation with disease incidence, but tended to increase and decrease a few quarters earlier than the disease incidence. Analyses of the associations between disease incidence and each of the cereal grains maize, wheat and barley, as well as the association between incidence and the primary variable comprising the latter three cereal types, were performed. Season and dietary animal proteins modified the associations between disease incidence and each of the feed variables. At above-median levels of animal proteins, significant associations were restricted to the primary variable, barley and maize, during the cold season. At below-median levels of animal proteins, the primary variable, barley and wheat, was associated with disease incidence. Above-median levels of maize protected against necrotic enteritis, whereas barley and wheat were risk factors.

The Influence of Diet on Necrotic Enteritis in Broiler Chickens

Necrotic enteritis was reproduced in broiler chickens by mixing cultures of Clostridium perfringens in the feed. Mortality due to necrotic enteritis was higher among chickens fed rations based on wheat, rye, barley, and oat groats than among chickens fed corn-based rations. Addition of pentosanase to a wheat-based diet did not affect the level of mortality due to necrotic enteritis. Addition of pectin and guar gum to different rations severely reduced growth rate and eliminated necrotic enteritis from test birds. Addition of glucose to a corn-based diet caused a small increase in mortality due to necrotic enteritis.

Effects of Eimeria brunetti Infection and Dietary Zinc on Experimental Induction of Necrotic Enteritis in Broiler Chickens

Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis. Inoculation with sporulated E. brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens. Feed was supplemented with zinc at 1000 ppm. Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc. Coccidial lesion scores were also highest in that group. Birds infected with E. brunetti and C. perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E. brunetti only, regardless of zinc supplementation. Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation. Zinc was tested for effects of alpha toxin production in vitro. In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin. Addition of zinc partly protected the toxin from the action of trypsin.

In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens

Viable Clostridium perfringens ranging from 10(5) to 10(8) g-1 was detected in all of 88 intestinal content specimens of necrotic enteritis in broiler chickens. In vitro lecithinase activity and sensitivity to 22 antimicrobial agents were determined for the 88 isolates. The activities of lecithinase in the culture filtrate of isolates were estimated to be 0.5 to 4.0 AE ml-1 as alpha-antitoxin equivalent. With reference to antimicrobial activity penicillins and cephazolin showed excellent activity and no resistance; peptides, of the agents used as growth promoters, showed that all except bacitracin had low minimum inhibitory concentration levels (1.6 micrograms ml-1 or less) against this organism; polyethers of monensin, salinomycin and lasalocid were generally adequate in low concentrations while there was a high level of resistance to three tetracyclines in 90 per cent of the strains and all isolates were insusceptible to streptomycin of the aminoglycoside antibiotics.