Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks

Historical data of necrotic enteritis incidence and carcass condemnations owing to liver lesions in south-eastern Norway during 1978-1998 revealed covariance in the occurrence of the two variables. In histological examination during the first half of 1998, 33 of 45 sampled carcasses condemned owing to liver lesions showed histopathological changes consistent with earlier described Clostridium perfringens-associated hepatitis. The results suggest that the occurrence of necrotic enteritis in broiler flocks may be monitored by using meat inspection data on liver lesions.     

Antimicrobial activity of essential oils and structurally related synthetic food additives towards Clostridium perfringens

Aims:  To assess the potential of essential oils and structurally related synthetic food additives in inhibiting the growth of Clostridium perfringens for the control of necrotic enteritis in chickens.
Methods and Results:  The antimicrobial activity of essential oils/compounds was measured by determining the inhibition of bacterial growth. Thirty-three of 66 oils/compounds exhibited ≥80% inhibition. Seven with the highest potency were further studied. The oils/compounds had MIC₉₅ values between 167 and 425  μ g ml⁻¹. Most of them were tolerant to low pH (2·0) and exhibited minor or no inhibition of Lactobacillus isolates from the chicken intestine. When mixed with chicken ileal digesta, the oils/compounds retained their efficacy against C . perfringens , but had little effect on the total number of lactobacilli and anaerobic bacteria in the digesta.
Conclusions:  Some essential oils/compounds demonstrated good potential in controlling C . perfringens .
Significance and Impact of the Study:  This study has identified candidates of essential oils/compounds for in vivo studies for the control of necrotic enteritis in chickens.     

An observation of Clostridium perfringens in Greater Sage-Grouse

Mortality due to infectious diseases is seldom reported in the Greater Sage-Grouse (Centrocercus urophasianus). A case of necrotic enteritis associated with Clostridium perfringens type A is described in a free-ranging adult male sage-grouse in eastern Oregon. Clostridial enteritis is known to cause outbreaks of mortality in various domestic and wild birds, and should be considered as a potential cause of mortality in sage-grouse populations.     

The ability of disease and non-disease producing strains of Clostridium perfringens from chickens to adhere to extracellular matrix molecules and Caco-2 cells

Clostridium perfringens is a major enteric pathogen that is responsible for causing necrotic enteritis of poultry. The ability to adhere to the host’s intestinal epithelium and to extracellular matrix molecules (ECMM) in the gut, are strategies used by numerous bacterial enteropathogens, however, C. perfringens has received comparatively little attention in this respect. The present study investigated sixteen type A C. perfringens isolates from chickens, with varying disease producing ability with respect to necrotic enteritis in chickens, for their ability to adhere to nine different extracellular matrix molecules (ECMM) and to the intestinal epithelial cell line Caco-2. C. perfringens strains were able to bind to ECMMs and there was strain variation. Strains of C. perfringens that produced severe disease, were capable of binding to collagen type III, IV and V, fibrinogen, laminin and vitronectin at higher levels than less severe disease producing strains, suggesting that the ability to adhere to ECMMs might enhance virulence with respect to induction of necrotic enteritis. In addition, severe disease producing strains also bound better to collagen type III and IV and fibrinogen, than non-disease producing strains. The present study also showed that some strains of C. perfringens possessed the ability to adhere to Caco-2 cells; however no relationship was found between the ability to adhere to Caco-2 cells and disease producing ability.     

Bacillus amyloliquefaciens BLCC1-0238 Alone or in Combination with Mannan-Oligosaccharides Alleviates Subclinical Necrotic Enteritis in Broilers

Probiotics and Antimicrobial Proteins - Subclinical necrotic enteritis (SNE) is a severe intestinal disease in broilers which brings huge economic losses to poultry industry. Herein, the effects of...     

Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

A Recombinant Probiotic, <Emphasis Type="Italic">Lactobacillus casei,</Emphasis> Expressing the <Emphasis Type="Italic">Clostridium perfringens</Emphasis> α-toxoid,as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa _247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.     

Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10² CFU/g of ileal material, but only about 10⁴ CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis isolated from the gastrointestinal tracts of healthy chickens

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121 degrees C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37 degrees C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.     

Inhibition of Clostridium perfringens by a Novel Strain of Bacillus subtilis Isolated from the Gastrointestinal Tracts of Healthy Chickens

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121°C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37°C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.     

Parasitic enteritis in superb lyrebirds (Menura novaehollandiae)

Ten free-living superb lyrebirds (Menura novaehollandiae) from forest habitat in southern Victoria, Australia were examined at necropsy over a 10 yr period. The acanthocephalan Plagiorhynchus menurae was identified in two lyrebirds from forest habitat in southern Victoria, Australia. There was necrotic enteritis in the duodenum associated with the acanthocephalans, with secondary bacterial involvement. The lesions probably resulted in the observed emaciation and debilitation of the birds. Probably the forest-floor habitat and insect diet of lyrebirds exposes them to these infections.     

Cooperativity of peptidoglycan synthases active in bacterial cell elongation

Clostridium perfringens possesses at least two functional quorum sensing (QS) systems, i.e. an Agr-like system and a LuxS-dependent AI-2 system. Both of those QS systems can reportedly control in vitro toxin production by C. perfringens but their importance for virulence has not been evaluated. Therefore, the current study assessed whether these QS systems might regulate the pathogenicity of CN3685, a C. perfringens type C strain. Since type C isolates cause both haemorrhagic necrotic enteritis and fatal enterotoxemias (where toxins produced in the intestines are absorbed into the circulation to target other internal organs), the ability of isogenic agrB or luxS mutants to cause necrotizing enteritis in rabbit small intestinal loops or enterotoxemic lethality in mice was evaluated. Results obtained strongly suggest that the Agr-like QS system, but not the LuxS-dependent AI-2 QS system, is required for CN3685 to cause haemorrhagic necrotizing enteritis, apparently because the Agr-like system regulates the production of beta toxin, which is essential for causing this pathology. The Agr-like system, but not the LuxS-mediated AI-2 system, was also important for CN3685 to cause fatal enterotoxemia. These results provide the first direct evidence supporting a role for any QS system in clostridial infections.     

Differential Responses of Cecal Microbiota to Fishmeal,Eimeria and Clostridium perfringens in a Necrotic Enteritis Challenge Model in Chickens

Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for progressing towards clinical symptoms of necrotic enteritis.     

Quantification of cell proliferation and alpha-toxin gene expression of Clostridium perfringens in 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.     

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.     

In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production

AIMS: Clostridium perfringens type A causes both clinical and subclinical forms of necrotic enteritis in domestic avian species. In this study the inhibitory effect of hen egg white lysozyme on the vegetative form of Cl. perfringens type A and the production of alpha-toxin in vitro was investigated. METHODS AND RESULTS: A micro-broth dilution assay was used to evaluate the minimal inhibitory concentrations (MIC) of lysozyme against three clinical isolates of Cl. perfringens type A in 96-well microtitre plates. The MIC of lysozyme against Cl. perfringens isolates was found to be 156 microg ml(-1). Scanning electron micrographs of the cells treated with 100 microg ml(-1) of lysozyme revealed extensive cell wall damage. A quantitative sandwich ELISA for alpha-toxin produced by Cl. perfringens was developed based on a commercial ELISA kit allowing only qualitative detection. Addition of 50 microg ml(-1) of lysozyme did not inhibit the growth of Cl. perfringens but significantly inhibited the toxin production. CONCLUSIONS: Lysozyme inhibited the growth of Cl. perfringens type A at 156 microg ml(-1). At sublethal levels, lysozyme was able to inhibit the alpha-toxin production. SIGNIFICANCE AND IMPACT OF STUDY: Inhibition of Cl. perfringens type A and its alpha-toxin production by hen egg white lysozyme had never previously been reported. By inhibiting this avian pathogen and its toxin production, lysozyme showed potential for use in the treatment and prevention of necrotic enteritis and other Cl. perfringens type A related animal diseases.     

Clostridium perfringens in retail chicken

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% “atypical” genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.     

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.     

Real-time PCR assay for Clostridium perfringens in broiler chickens in a challenge model of necrotic enteritis

We compared ileal Clostridium perfringens quantification results produced by real-time PCR and culture-based methods in broiler chickens in a challenge model of necrotic enteritis. Assessment of the relative standard deviations (RSDs) revealed that the real-time PCR assay generated a smaller standard deviation and thus was more precise than the culture-based method. Linear regression analysis indicated that the bacterial counts of these two methods were highly correlated (R(2) = 0.845). We suggest that real-time PCR could be a replacement of the culture method for quantifying C. perfringens in the intestinal tracts of broiler chickens.