Genetic relatedness and netB prevalence among environmental Clostridium perfringens strains associated with a broiler flock affected by mild necrotic enteritis

In a previous study we investigated pulsed-field gel electrophoresis (PFGE) genotype diversity and prevalence of the netB toxin gene in Clostridium perfringens (CP) isolates recovered from a broiler flock (flock 1) affected by necrotic enteritis (NE). In this follow-up work, we examined samples collected before placement of flock 1, to see if NE during rearing could be traced back to the cleaned and empty building or the day-old chicks. Litter from the next flock in the same building (flock 2) was also examined. We detected 25 different PFGE genotypes, five of which were found only in litter from flock 2. Six genotypes which had been found in flock 1 during rearing were detected in samples collected before placement. NetB positive isolates belonging to two of these genotypes had been recovered from NE lesions during rearing, suggesting that virulent strains were transmitted from the cleaned and disinfected broiler house. NetB frequency among isolates from the empty building was 45%, indicating that netB positive strains were prevalent in a building that previously had housed a healthy flock offered in-feed narasin (flock 0). NetB frequency among isolates from litter used by flock 2 was 22%, indicating that netB positive strains were present in the environment of a 14-days-old healthy flock offered in-feed narasin. Two prevalent genotypes were consistently either netB negative or netB positive. However, the presence of genotypes represented by both negative and positive isolates may suggest that the gene can spread horizontally among different CP strains.     

Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin

A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.     

Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers

Since the ban on growth-promoting antibiotics in animal feed in the European Union, necrotic enteritis has become a major cause of mortality in broiler chickens. Despite the importance of the disease, the pathogenesis is still not completely understood. In the current study, Clostridium perfringens strains isolated from healthy flocks and isolates from outbreaks of necrotic enteritis were evaluated for the ability to cause gut necrosis in an intestinal loop model in laying hens and in an experimental infection model in broilers. High, intermediate and low alpha toxin producing strains were chosen from each isolation source. Only the isolates from field outbreaks induced necrotic gut lesions, independent of the amount of alpha toxin produced in vitro. It was also shown that alpha toxin producing isolates from calf hemorrhagic enteritis cases were not able to induce necrotic enteritis in poultry. These results suggest the presence of host specific virulence factors in C. perfringens strains, isolated from chickens with intestinal necrotic enteritis lesions.     

Hemorrhagic enteritis associated with Clostridium perfringens type A in a dog

A female Shetland sheep dog died suddenly with hemorrhagic diarrhea and vomitting, and was examined pathologically and microbiologically. Gross pathological change was restricted to the intestinal tract. The intestine contained watery, blood-stained fluid. Histopathologically, the principal intestinal lesion was superficial mucosal hemorrhagic necrosis at the jejunoileum. Many Gram-positive bacilli were found adhering to the necrotic mucosal surface in parts of the intestinal tract. Clostridium perfringens in pure culture were isolated from jejunal contents by anaerobic culture. These results suggested that the typical lesion of this case coincided with canine hemorrhagic enteritis and enterotoxemia due to C. perfringens infection could be the cause of sudden death.     

Haemorrhagic necrotising enteritis in foals associated with Clostridium perfringens

Two foals aged 35 and 48 h from 2 Thoroughbred studs died several hours after developing clinical signs of depression, severe haemorrhagic diarrhoea and dehydration. Both foals had an acute haemorrhagic enteritis extending from the anterior jejunum to the terminal ileum which was characterised histologically by villus necrosis. Necrotic villi were surrounded by large numbers of rod-shaped Gram positive bacteria. Clostridium perfringens was recovered from the intestines of both foals and the isolates were considered to be C. perfringens type C. Other cases of diarrhoea were also observed in foals of the same age on these 2 studs, but the aetiology of these was not determined.     

Necrotizing enteritis in suckling pigs (Clostridium perfringens type C enterotoxemia). II. Toxin formation, heat and drug resistance of Clostridium perfringens strains isolated from suckling pigs and broilers with necrotizing enteritis]

Nineteen Clostridium perfringens Type C strains and ten foreign control strains of subtypes C1, C3, and C4 were tested for their toxin formation and spore resistance to heat. The 19 Type C strains had been isolated from unweaned piglets in the context of necrotising enteritis outbreaks in the GDR. The Clostridium perfringens Type C strains formed beta-toxin, but they failed to form epsilon-toxin or gamma-toxin, alpha-toxin was successfully recorded from 15 of the 19 strains tested from unweaned piglets. The minor-lethal toxin fractions were also tested, with delta-toxin being recorded from all strains, non-alpha-delta-theta-toxin from six, theta-toxin from five, and K-toxin from one. Tests for delta-toxin, lambda-toxin, and mu-toxin were negative. The Clostridium perfringens Type C strains isolated in the GDR from unweaned piglets with necrotising enteritis were, basically, identical with those described in Denmark by von Hogh (1967) with regard to toxin formation. Clostridium perfringens strains cultured in broilers with necrotising enteritis were characterised by regular toxin production in the context of alpha, theta, delta as well as non-alpha-delta-theta. They failed to form beta, epsilon, gamma and lambda, while mu-toxin was formed by them quite irregularly. They, consequently, are Type A strains. Resistance to chloramphenicol and/or oxytetracycline was exhibited by 78.5 per cent of 237 tested Clostridium perfringens strains which had been isolated from unweaned piglets and broilers with necrotosing enteritis. Multiple resistance was recorded from 33.9 per cent. All strains were susceptible to penicillin, nitrofurantoin, and erythromycin.     

Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis

The present study examines the responses of broiler chickens to oral administration of Clostridium perfringens freshly isolated from field cases of necrotic enteritis (NE). The challenge studies included long-term exposure and short-term exposure, factored in with dietary and management variables including high levels of dietary components such as fish meal, meat meal, abrupt change of feed, and fasting. In the long-term exposure trials, the birds were orally inoculated daily, with 1 ml (1.0 or 2 x 10(8) CFU/ml) of an overnight culture of C. perfringens for 7 days. Short-term exposure trials involved challenge with 1 ml (3 x 10(10) CFU/ml) administered as a single dose. The responses of broilers to orally administered C. perfingens under laboratory controlled conditions are presented and discussed in the context of authentic field cases of necrotic enteritis. None of the challenge trials produced overt clinical signs of NE and there were no mortalities associated with oral exposure to high doses of C. perfringens. However, many of the challenged birds showed distinctly pronounced pathological changes in the intestinal tissue. On gross examination the responses in birds challenged orally with C. perfringens could be placed into two categories: (1) no apparent pathological changes in the intestinal tissue and (2) sub-clinical inflammatory responses with focal, multi-focal, locally extensive, or disseminated distribution throughout various sections of duodenum, jejunum, ileum, and ceca. In birds that responded with intestinal lesions, hyperemia and occasional hemorrhages were the main gross changes. In some birds, the mucosa was covered with a brownish material, but typically, the mucosa was lined by yellow or greenish, loosely adherent material. Mild gross changes were seen in some control birds, but both qualitatively and quantitatively, the lesions were distinctly more pronounced in the challenged birds. Upon histological examination, none of the experimentally exposed birds showed overt mucosal necrosis typical of field cases of NE, but typically the lamina propria was hyperemic and infiltrated with numerous inflammatory cells. Most significant changes were seen at the interface of the basal domain of enterocytes and lamina propria. Multifocally, these areas were extensively edematous, allowing for the substantial disturbance of the structural integrity between the lamina propria and the enterocytes. The lesions observed in the present study were consistently reproduced in all of our challenge trials, hence these responses may signify newly emerging patterns of sub-clinical enteric disorders in contemporary strains of poultry. The pathological changes observed in broilers challenged orally with C. perfringens in the present study, differ significantly from those reported previously, and must be clearly differentiated from those described in cases of NE or ulcerative enteritis. Although no overt necrosis of the intestinal mucosa typical of field cases of NE were observed in the present study, the birds challenged with C. perfringens showed strong inflammatory reaction to the introduced pathogens. The distinct features of the microscopic lesions were changes involving apparently normal enterocytes at the interface of the basal domain of villar epithelia and lamina propria. Although the pathological changes in the intestinal tissues observed in our trials appear to be rather subtle when compared to field cases of NE, the nature of these lesions suggest a significant negative effect on the digestive physiology of intestinal mucosa.     

甘草总黄酮对鸡产气荚膜梭菌坏死性肠炎的治疗作用

选择具有显著抗炎效果的甘草总黄酮为受试化合物,考察其抗产气荚膜梭菌所致鸡坏死性肠炎的效果,为甘草总黄酮在坏死性肠炎的治疗应用提供依据。选择AA^+肉鸡,经口给予产气荚膜梭菌ATCC13124菌悬液建立肉仔鸡坏死性肠炎模型,之后使用甘草总黄酮对造模成功的肉鸡进行治疗,通过剖检考察肉鸡肠道的病变情况、病变评分、日增重及器官指数等指标以评估甘草总黄酮对坏死性肠炎的治疗效果。结果显示,通过经口攻菌成功建立了肉仔鸡坏死性肠炎模型;剖检发现,经甘草总黄酮治疗的模型鸡其肠道病变程度明显缓解,病变损伤评分显著低于感染组,炎性因子含量显著降低;治疗组肉鸡日增质量及器官指数与健康肉鸡差异不显著。结果表明,甘草总黄酮能够缓解产气荚膜梭菌所致坏死性肠炎对肉鸡肠道的损伤,具有作为抗生素替代品治疗坏死性肠炎的潜力。     

Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks

We inoculated broiler chicks with mixtures of Clostridium perfringens strains to investigate the single strain dominance observed in natural cases of necrotic enteritis (NE) [Nauerby, B., Pedersen, K., Madsen, M., 2003. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 94, 257-266]. Pre-inoculation bacteriologic culture of chick intestines yielded up to six pulsed-field gel electrophoresis (PFGE) types of C. perfringens. Birds developed typical NE lesions in response to administration (2x per day for 4 days) of a combined inoculum comprising one NE strain (JGS4143, PFGE pattern 8) and four non-NE strains (from piglet necrotizing enteritis, chicken normal flora, human gas gangrene, and bovine neonatal enteritis). After inoculation commenced, only the NE strain was recovered through the first post-inoculation day, in spite of intense efforts to recover pre-challenge flora strains and the other challenge strains. Thereafter, pre-inoculation and previously undetected PFGE types were found, and JGS4143 became undetectable. Birds inoculated simultaneously with five NE strains (from disease in chickens or turkeys, and including JGS4143) also developed lesions, but again only JGS4143 was recovered through the 1st day post-challenge. At that time, birds began to be repopulated with pre-challenge PFGE types. Two NE strains (JGS4143 and JGS4064) produced bacteriocins, which inhibited each other and normal flora strains (n=17), while normal flora strains inhibited neither NE strains nor each other. Thus, it appears that naturally occurring dominance of the gut by NE strains can be reproduced experimentally. Bacteriocins directed against normal flora could possibly provide the necessary advantage, although inhibition of one NE strain by another suggests that other factors may be partially or completely responsible for the dominance.     

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.     

The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review

Necrotic enteritis (NE) is one of the most important enteric diseases in poultry and is a high cost to the industry worldwide. It is caused by avian-specific, Necrotic Enteritis Beta toxin (NetB)-producing, strains of Clostridium perfringens that also possess in common other virulence-associated genes. In Europe the disease incidence has increased since the ban on in-feed “growth promoting” antibiotics. Because of this, many recent studies of NE have focused on finding different ways to control the disease, and on understanding its pathogenesis. Frustratingly, reproduction of the disease has proven impossible for some researchers. This review describes and discusses factors known to be important in reproducing the disease experimentally, as well as other considerations in reproducing the disease. The critical bacterial factor is the use of virulent, netB-positive, strains; virulence can be enhanced by using tpeL- positive strains and by the use of young rather than old broth cultures to increase toxin expression. Intestinal damaging factors, notably the use of concurrent or preceding coccidial infection, or administration of coccidial vaccines, combined with netB-positive C. perfringens administration, can also be used to induce NE. Nutritional factors, particularly feeding high percentage of cereals containing non-starch polysaccharides (NSP) (wheat, rye, and barley) enhance disease by increasing digesta viscosity, mucus production and bacterial growth. Animal proteins, especially fish meal, enhance C. perfringens proliferation and toxin production. Other factors are discussed that may affect outcome but for which evidence of their importance is lacking. The review compares the different challenge approaches; depending on the aim of particular studies, the different critical factors can be adjusted to affect the severity of the lesions induced. A standardized scoring system is proposed for international adoption based on gross rather than histopathological lesions; if universally adopted this will allow better comparison between studies done by different researchers. Also a scoring system is provided to assist decisions on humane euthanasia of sick birds.     

Sandwich ELISA detection of Clostridium perfringens cells and alpha-toxin from field cases of necrotic enteritis of poultry

Sandwich ELISAs (sELISAs) for the detection of Clostridium perfringens cells and alpha-toxin were developed and used to screen intestinal samples from normal broiler chickens and from clinical cases of necrotic enteritis. The assays clearly distinguished between the two sets of samples. The sELISA absorbance values from samples obtained from the majority of healthy birds were low and those from the majority of necrotic enteritis cases were high. Together, the assays provide a suitable test for the rapid screening for the diagnosis of necrotic enteritis in poultry.     

Optimized necrotic enteritis model producing clinical and subclinical infection of Clostridium perfringens in broiler chickens

In this study we assessed the roles of Eimeria infection and dietary manipulation (feeding a diet with a high level of fishmeal) in an Australian necrotic enteritis (NE) challenge model in broiler chickens. An experiment was designed to test the hypothesis that Eimeria infection and dietary manipulation, i.e., inclusion of fishmeal in the diet, are necessary to induce NE experimentally. The results showed that the combination of Eimeria administration and fishmeal feeding had a significant effect on induction of clinical and subclinical Clostridium perfringens infection. The majority of the mortality that occurred during the second week of the trial was due to an NE outbreak following the C. perfringens challenge. The mortality rate of the birds was 12.00% for the high-fishmeal (HFM; 500 g/kg) group and 9.33% for the low-fishmeal (LFM; 250 g/kg) group when the birds were subjected to C. perfringens and Eimeria. Fishmeal alone did not induce significant mortality in birds challenged only with C. perfringens but showed a significantly higher C. perfringens count than the non-fishmeal (NFM) control group. Eimeria administration had a significant effect on NE-related mortality but did not have an effect on the C. perfringens count. In accordance with the time course of bird mortality, it can be determined that of the 3 successive days of oral gavage with C. perfringens, the first inoculation was essential for inducing NE, but the third had no additional effect on NE-related mortality. Also, reducing the fishmeal level from 500 to 250 g/kg had no negative impact on the reproducibility of the model. It may be concluded that NE can be consistently induced under experimental conditions by feeding broilers a diet containing 250 g/kg fishmeal, using a single inoculation with low numbers of Eimeria, administering one or two oral C. perfringens inoculations, and maintaining appropriate ambient temperatures and diets.     

Molecular detection and characterization of Clostridium perfringens toxin genes causing necrotic enteritis in broiler chickens

Tropical Animal Health and Production - A total of 464 samples comprising of cloacal swabs from necrotic enteritis suspected live birds (191), intestinal scrapings from dead birds with symptoms of...     

Changes in the caecal microflora of chickens following Clostridium perfringens challenge to induce necrotic enteritis

Necrotic enteritis is a disease of considerable economic importance to the global poultry industry. Clostridium perfringens has long been recognised as the etiological agent of the disease. However, disease initiation and progression is complex and appears to be precipitated by a range of predisposing factors. The present study investigated microbial interactions in the caecum of birds challenged with C. perfringens that developed necrotic enteritis. Bacterial populations of healthy and diseased birds, across two independent animal trials, were characterised by pyrosequencing of the V1-V3 region of 16S rRNA genes. Significant changes in the microbiota of infected birds were detected. Most of the affected bacterial species, including a number of butyrate producers, were reduced in abundance in infected birds compared to uninfected controls and a number of phylotypes, classified as Weissella species, were also more abundant in healthy birds. Conversely, some bacterial groups were more abundant in the C. perfringens-infected birds, for example, members of an unclassified order of Mollicutes showed a 3.7-fold increase in abundance in infected birds. Representative sequences from this novel order shared 99% identity with sequences previously detected in intestinal microbiota of chickens and humans, and have previously been shown to be represented in a number of samples originating from irritable bowel syndrome disease patients. We speculate that these newly identified perturbations in the composition of caecal microflora may play a role in the development and manifestation of necrotic enteritis.     

Vegetative Bacillus amyloliquefaciens cells do not confer protection against necrotic enteritis in broilers despite high antibacterial activity of its supernatant against Clostridium perfringens in vitro

1. In this study, the effect of Bacillus amyloliquefaciens on Clostridium perfringens was tested in vitro and in vivo.

2. Using an agar well diffusion assay, the inhibitory activity of B. amyloliquefaciens supernatant was analysed against a large collection of netB-positive and netB-negative C. perfringens strains. Although strong growth inhibiting activity was detected against all C. perfringens isolates, it was significantly higher against virulent netB-positive C. perfringens strains compared with avirulent netB-negative isolates.

3. Subsequently, the efficacy of in-feed administration of lyophilised vegetative cells of B. amyloliquefaciens to prevent necrotic enteritis was tested in vivo using an established experimental infection model in broilers. Ross 308 broilers received either B. amyloliquefaciens supplemented or unsupplemented feed throughout the experiment. No significant differences could be detected between the untreated positive control group and the B. amyloliquefaciens treated group in body weight, the number of chickens that developed necrotic lesions and in pathological lesion scores.

4. These results demonstrate that despite its substantial inhibitory activity in vitro, lyophilised vegetative B. amyloliquefaciens cells had no beneficial effect against necrotic enteritis in the in vivo model used here.

     

Antimicrobial resistance in Clostridium perfringens isolates from broilers in Belgium

The antimicrobial susceptibility of Clostridium perfringens strains isolated from Belgian broilers between May and September 2007 was investigated. All 39 tested isolates were sensitive to enrofloxacin, erythromycin, tylosin, florfenicol and bacitracin. Twenty-six (66%) and 24 (61%) out of the 39 tested isolates showed acquired resistance to tetracycline and lincomycin, respectively. The C. perfringens isolates were also screened by PCR for the presence of the resistance genes tet(K), tet(L), tet(M), tetB(P), tet(O), tet(W), lnu(A) and lnu(B). In 22/26 tetracycline resistant strains and 7/24 lincomycin resistant strains, resistance could be attributed to one or more of these genes. An extended frequency distribution range of MICs was seen for ampicillin. These data are consistent with data derived from studies carried out in 1980 and in 2004, indicating that no changes in antimicrobial resistance patterns have taken place during time in C. perfringens isolates from broilers in Belgium.