NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands

Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.     

Progress and problems in vaccination against necrotic enteritis in broiler chickens

Necrotic enteritis in broilers is caused by Clostridium perfringens type A strains that produce the NetB toxin. Necrotic enteritis is one of the gastrointestinal diseases in poultry that has gained worldwide importance during the last decade due to efforts to improve broiler performance. Prevention strategies include avoiding predisposing factors, such as coccidiosis, and in-feed supplementation with a variety of feed additives. However, vaccination with modified toxin or other secreted immunogenic proteins seems a logical preventive tool for protection against a toxin-producing bacterium. Formalin-inactivated crude supernatant has been used initially for vaccination. Several studies have been carried out recently to identify the most important immunogenic and protective proteins that can be used for vaccination. These include the NetB toxin, as well as a number of other proteins. There is evidence that immunization with single proteins is not protective against severe challenge and that combinations of different antigens are needed. Most published studies have used multiple dosage vaccination regimens that are not relevant for practical use in the broiler industry. Single vaccination regimens for 1-day-old chicks appear to be non-protective. This review describes the history of vaccination strategies against necrotic enteritis in broilers and gives an update on future vaccination strategies that are applicable in the field. These may include breeder hen vaccination, in ovo vaccination and live attenuated vectors to be used in feed or in drinking water.     

Temporary feed restriction partially protects broilers from necrotic enteritis

The objective of this study was to investigate the effect of feed restriction on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chicks. To induce subclinical necrotic enteritis, an experimental challenge model using a specific diet formulation, Gumboro vaccination, oral inoculation of broilers with a 10-fold dose of attenuated anticoccidial vaccine and multiple oral inoculations with a specific strain of Clostridium perfringens was adopted. Two hundred and forty 1-day-old Cobb 500 broilers were randomly allocated to four groups: feed restricted, challenged, both feed restricted and challenged, and negative control. At 21, 22, 23 and 24 days of age, the intestines, gizzard and liver were collected from 15 birds in each group and scored for gross lesions. The intestinal digesta was collected for pH and viscosity determination. One caecum from each bird was taken for microbiological analysis. The application of feed restriction in birds challenged with C. perfringens reduced the necrotic enteritis lesion score significantly (P ≤ 0.05) and feed restriction significantly reduced (P ≤ 0.05) pH in the small intestine, the viscosity of the jejunum digesta as well as the C. perfringens counts in the caeca compared with the controls. In conclusion, feed restriction of broilers has a positive effect on the intestinal ecosystem and a significant protective effect against necrotic enteritis in the subclinical experimental model.     

Maternal vaccination against subclinical necrotic enteritis in broilers

The inclusion of antibacterial feed additives has until now been the major strategy for controlling Clostridium perfringens-associated necrotic enteritis in broilers. In the present study, the effect of maternal immunization against the disease was examined. Broiler breeder hens were injected intramuscularly with candidate vaccines based on C. perfringens type A and type C toxoids adjuvanted with aluminium hydroxide. Vaccination resulted in a strong serum immunoglobulin G response to C. perfringens alpha-toxin in parent hens, and specific antibodies were transferred to their progeny. Subclinical necrotic enteritis in broilers was induced under field conditions or in a disease model, and the occurrence of specific enteric and hepatic lesions was evaluated in randomly selected birds. In three experiments, estimates of odds ratio for developing such lesions were 0.23, 0.33 and 0.56 in maternally toxoid C-immunized broilers compared with non-immunized controls. In toxoid A-immunized birds, odds ratios were estimated at 0.41, 0.61 and 0.63. From these results, immunoprophylaxis seems to be an interesting alternative for the control of necrotic enteritis in broilers.     

Clostridial co-infection episodes in commercial laying hens

The present report describes two outbreaks of serious enteritis in commercial laying hens where Clostridium perfringens and Clostridium colinum were simultaneously detected. At the age of 44 and 31 weeks, two laying hen flocks showed an increase of the mortality rate and a worsening of productive performance. Post-mortem examination revealed intestinal necrotic-haemorrhagic ulcerations and hepatic focal necrosis. The bacteriological examination yielded the isolation of C. colinum and C. perfringens toxin type A, NetB positive. In one outbreak, C. colinum was detected also by polymerase chain reaction in all the intestines of affected birds. In laying hens, C. colinum has never been isolated but only suspected as the causative agent of a slight enteric disease called duodenal focal necrosis. The present case report was characterized by severe enteritis presumably due to the synergistic effect of C. colinum and C. perfringens.     

Diagnosing Clostridium perfringens-associated necrotic enteritis in broiler flocks by an immunoglobulin G anti-alpha-toxin enzyme-linked immunosorbent assay

The tools available for monitoring necrotic enteritis caused by Clostridium perfringens in broiler chickens have been limited, particularly for identifying subclinical disease. In this study, a modified enzyme-linked immunosorbent assay was used to quantify levels of specific immunoglobulin G to C. perfringens alpha-toxin in serum from broilers. We found significantly higher antibody levels in broilers with a history of subclinical necrotic enteritis compared with a zinc-bacitracin-treated group with a low level of gut lesions. Furthermore, in 4.5-week-old commercial broiler flocks, there was an association between the occurrence of C. perfringens-associated hepatitis at slaughter and the immune response to alpha-toxin. Practical solutions for defining cut-off levels for positive serum samples at individual and flock levels are proposed, and were found to be useful on a set of samples available from flocks with different histories regarding the occurrence of C. perfringens-associated disease. This serological approach seems promising as a diagnostic tool in research and disease monitoring regarding C. perfringens-associated disease.     

Day-of-hatch vaccination is not protective against necrotic enteritis in broiler chickens

Necrotic enteritis, caused by netB toxin-producing Clostridium perfringens type A, is an important disease in broiler chickens worldwide. Earlier attempts to prevent necrotic enteritis by vaccination have not sufficiently taken into account the practical limitations of broiler vaccination. In most published studies on vaccination against necrotic enteritis, multiple doses at different ages are administered, which is not practical for broilers. The aim of this study was to compare the efficacy of subcutaneous single vaccination at day 1 or day 3 and double vaccination at day 3 and day 12, using crude supernatant containing active toxin or formaldehyde-inactivated supernatant (toxoid) of a netB-positive C. perfringens strain in a subclinical necrotic enteritis model. Double vaccination with crude supernatant resulted in a significant decrease in the number of chickens with necrotic enteritis lesions. The efficacy of vaccination using toxoid was lower compared with crude supernatant. Single vaccination with crude supernatant at day 3 resulted in significant protection, while vaccination of 1-day-old chickens with crude supernatant or toxoid, as envisaged for practical field application, did not induce protection.     

Toxinotyping of necrotic enteritis-producing and commensal isolates of Clostridium perfringens from chickens fed organic diets

The present study determined the effect of Clostridium perfringens isolates taken from necrotic enteritis (NE) outbreaks on organic farms in a NE virulence testing model. Thirteen strains were isolated in the course of the study. Six C. perfringens field isolates were taken from a naturally occurring NE outbreak on an organic farm. Polymerase chain reaction toxinotyping was used to establish C. perfringens strains, as well as to create a toxin profile. All field isolates were found to be type A and positive for alpha, beta-2 and netB toxin genes. During the NE virulence model, digesta samples were collected before oral inoculation to define the C. perfringens found as part of the natural flora. Three of the five natural flora isolates were found to be C. perfringens type E while the other two isolates were type A; only four of five isolates were positive for either netB or beta-2 toxin genes. Two isolates collected after inoculation were C. perfringens type A positive for cpb2 and netB. All isolates were tested positive for the quorum-sensing-related gene luxS, regardless of the strain source. The presence of luxS, alpha, netB and beta-2 toxin genes seems not to be a determinant of the disease as they were present in isolates from both outbreak birds as well as healthy and pre-inoculated birds. The C. perfringens field isolates induced mild NE lesions in one-half of the birds during the challenge study. Other mechanisms must play a role in the development of the disease beyond toxinotype, potentially including intestinal ecology and health, which would account for acute disease as seen in the field outbreak.     

The effect of commonly used anticoccidials and antibiotics in a subclinical necrotic enteritis model

Necrotic enteritis poses an important health risk to broilers. The ionophore anticoccidials lasalocid, salinomycin, maduramicin, narasin and a combination of narasin and nicarbazin were tested in feed for their prophylactic effect on the incidence of necrotic enteritis in a subclinical experimental infection model that uses coccidia as a predisposing factor. In addition, drinking water medication with the antibiotics amoxicillin, tylosin and lincomycin was evaluated as curative treatment in the same experimental model. The minimal inhibitory concentrations (MICs) of all antibiotics and anticoccidials were determined in vitro against 51 Clostridium perfringens strains isolated from broilers. The strains examined appeared uniformly susceptible to lasalocid, maduramicin, narasin, salinomycin, amoxicillin and tylosin, whereas an extended frequency distribution range of MICs for lincomycin was seen, indicating acquired resistance in 36 isolates in the higher range of MICs. Nicarbazin did not inhibit the in vitro growth of the C. perfringens strains even at a concentration of 128 µg/ml. Supplementation of the diet from day 1 onwards with lasalocid, salinomycin, narasin or maduramicin led to a reduction in birds with necrotic enteritis lesions as compared with the non-medicated infected control group. A combination product of narasin and nicarbazin had no significant protective effect. Treatment with amoxicillin, lincomycin and tylosin completely stopped the development of necrotic lesions.     

A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks

Necrotic enteritis (NE) is a major problem in antibiotic-free (ABF) chicken flocks and specific strains of Clostridium perfringens are known to induce NE. The objective of this study was to develop a chicken intestinal ligated loop model in order to compare the virulence of various C. perfringens strains recovered from consecutive ABF flocks with and without NE. Intestinal loops were surgically prepared in 10 anaesthetized specific-pathogen-free chickens and alternately inoculated with C. perfringens isolates or brain heart infusion (BHI) media. Histological lesion scoring was performed for each loop. All strains from NE-affected flocks induced histological lesions compatible with NE whereas inoculation of loops with a commensal C. perfringens strain or BHI did not. Among inoculated strains, CP0994 (netB-positive and cpb2-positive) and CP-2003-1256 (netB-positive) demonstrated mean histological lesion scores significantly higher (P?netB-negative and cpb2-positive) induced intestinal lesions without significantly higher scores. In loops where villi were colonized by Gram-positive rods, significantly higher (P?C. perfringens is a critical step in the pathogenesis of NE. Finally, we demonstrated the importance of controlling virulent C. perfringens strains in ABF chicken flocks as a highly virulent strain can be present in consecutive flocks with NE and possibly affect multiple flocks.     

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.     

An outbreak of gangrenous dermatitis in commercial broiler chickens

The present report describes an outbreak of gangrenous dermatitis (GD) infection in a commercial poultry farm in Delaware involving 34-day-old broiler chickens. In addition to obvious clinical signs, some GD-affected broilers also showed severe fibrino-necrotic enteritis and large numbers of Gram-positive rods in the necrotic tissue. Histopathological findings included haemorrhage, degeneration and necrosis of parenchymatous cells, especially of skin, muscle, and intestine. Immunofluorescence staining revealed Clostridium-like bacilli in the skin and the intestine. Both Clostridium perfringens and Clostridium septicum genomic sequences were identified by polymerase chain reaction in bacterial cultures isolated from the skin, muscle, and intestine, and in the frozen tissues from the GD-affected birds. Serological analysis demonstrated that both affected and clinically healthy birds from the same house had high serum antibody titres against C. perfringens, C. septicum, Eimeria, chick anaemia virus, and infectious bursal disease virus. These results are discussed in the context of the relationship between the different Clostridium spp. and the pathogenesis of GD.     

The effect of Artemisia annua on broiler performance,on intestinal microbiota and on the course of a Clostridium perfringens infection applying a necrotic enteritis disease model

The aerial parts of the plant Artemisia annua contain essential oils having antimicrobial properties against Clostridium perfringens Type A, the causal agent for necrotic enteritis in broilers. In two experiments, the influence of increasing dietary concentrations of dried A. annua leaves (0, 5, 10 and 20 g/kg) and n-hexane extract from fresh A. annua leaves (0, 125, 250 and 500 mg/kg) on broiler performance was investigated. Dried plant material decreased feed intake and body weight in a dose-dependent manner, and 10 and 20 g/kg diet tended to improve the feed conversion ratio. The n-hexane extract also reduced feed intake, but broiler weight tended to decrease only at the highest dietary concentration. The feed conversion ratio tended to improve when birds received 250 and 500 mg/kg n-hexane extract. In a third experiment, a necrotic enteritis disease model was applied to investigate the effect of the dietary addition of dried A. annua leaves (10 g/kg on top) or n-hexane extract of A. annua (250 mg/kg) on the severity of the disease in broilers. The addition of n-hexane extract reduced the intestinal C. perfringens numbers and the severity of the disease-related small intestinal lesions. Over the infection period from day 17 to day 27, birds supplemented with the n-hexane extract gained more weight than both the challenged control birds and birds receiving dried plant material. The results indicate that n-hexane extracts derived from A. annua can modulate the course of necrotic enteritis and compensate to a certain extent for the disease-associated weight losses.     

Non-soluble fibres and narasin reduce spontaneous gizzard erosion and ulceration in broiler chickens

The prevalence and severity of gizzard erosion and ulceration syndrome (GEU) in broilers is affected by a number of predisposing and preventive factors. Two broiler experiments with almost identical basal diets were conducted to investigate the effect of dietary oat hulls, access to litter and the antimicrobial compound narasin on GEU. The effects on particle size of duodenal digesta, ileal starch concentration, caecal Clostridium perfringens counts, necrotic enteritis and production performance were also examined. Oat hulls reduced GEU severity and starch levels in the ileum in both experiments. Access to litter reduced GEU scores when oat hulls were included in the feed. Access to litter also improved feed efficiency and reduced C. perfringens counts. Narasin reduced GEU severity when the feed was supplemented with oat hulls. Oat hulls were associated with improved feed efficiency in Experiment 1 and impaired feed efficiency in Experiment 2. The inconsistent effect of oat hulls on production performance appeared to be related to an association between oat hulls and high C. perfringens counts in Experiment 2; an association that was absent in Experiment 1. In general, oat hulls interacted with litter access and narasin in exerting a positive effect on gizzard health. However, the association between oat hulls and necrotic enteritis detected in Experiment 2 suggests that the positive effect of oat hulls on GEU occasionally may be outweighed by a negative effect on gut health. In conclusion, our findings demonstrate that the severity of GEU in broilers was influenced by non-soluble fibres and narasin.     

Safety and efficacy of a maternal vaccine for the passive protection of broiler chicks against necrotic enteritis

Necrotic enteritis is a potentially fatal multifactorial disease of chickens, which under commercial conditions is often associated with increased levels of mortality and reduced bird performance. The safety and efficacy of a Clostridium perfringens type A alpha-toxoid (Netvax?) formulated as an oil emulsion was investigated, following maternal immunization of broiler breeder hens, housed under commercial conditions, by the intramuscular route. A total of 11,234 hens were vaccinated across two integrated poultry sites. The vaccine was safe with no systemic reactions or adverse effects on bird performance detected. Vaccination resulted in a significant increase in anti-alpha toxin antibody in the hen that was maintained throughout the study, and subsequently transferred to their progeny throughout the laying period via egg yolk. Chicks hatched from eggs produced from vaccinated hens were shown to have reduced mortality specifically related to progeny flocks where gross gut lesions associated with necrotic enteritis were observed in control chicks. Further, whilst C. perfringens was isolated from control chicks with necrotic enteritis lesions, no such isolations were made at these time points from chicks from vaccinated hens. These results indicate that, under commercial conditions, maternal vaccination with Netvax? can help to control losses related to necrotic enteritis.     

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage,Production, Cytotoxic Contributions,and Trypsin Sensitivity

Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age.     

The effect of cold stress on the pathogenesis of necrotic enteritis in broiler chicks

Cold stress is a physical environmental stressor with significant effect on the poultry industry. The aim of the present study was to investigate the effect of cold stress as a predisposing factor for necrotic enteritis in broiler chicks. The experimental challenge model included an oral inoculation with 10-fold dose of attenuated anticoccidial vaccine and multiple oral inoculations with a specific strain of Clostridium perfringens. Birds were either challenged or not as described above, and either exposed or not to repeated cold stress (15°C for 12?h/day for 4 days). From each bird, intestinal gross lesions were scored and intestinal digesta pH and viscosity were measured. C. perfringens was counted in the caecum. The statistical analysis and evaluation of the experimental data revealed that the cold stress in challenged birds significantly increased the incidence and the severity of necrotic enteritis lesions (Ρ?≤?0.05), while causing no lesions in unchallenged birds. Moreover, the cold stress caused a significant increase (Ρ?≤?0.05) in the pH and C. perfringens counts in the caeca. The study provides evidence that cold stress increased the susceptibility to necrotic enteritis in a subclinical experimental model and thus should be regarded as a physical environmental stressor that could significantly affect the welfare, health and intestinal ecosystem of broiler chicks.     

Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.