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.     

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.     

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...     

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.     

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.     

Maternal immunization with vaccines containing recombinant NetB toxin partially protects progeny chickens from necrotic enteritis

Avian necrotic enteritis is a major economic and welfare issue throughout the global poultry industry and is caused by isolates of Clostridium perfringens that produce NetB toxin. Previously we have shown that birds directly vaccinated with inactivated C. perfringens type A culture supernatant (toxoid) combined with recombinant NetB (rNetB) protein were significantly protected from homologous and heterologous challenge. In the present study the protective effect of maternal immunization was examined. Broiler breeder hens were injected subcutaneously with genetically toxoided rNetB(S254L) alone, C. perfringens type A toxoid and toxoid combined with rNetB(S254L). Vaccination resulted in a strong serum immunoglobulin Y response to NetB in hens immunized with rNetB(S254L) formulations. Anti-NetB antibodies were transferred to the eggs and on into the hatched progeny. Subclinical necrotic enteritis was induced experimentally in the progeny and the occurrence of specific necrotic enteritis lesions evaluated. Birds derived from hens immunized with rNetB(S254L) combined with toxoid and challenged with a homologous strain (EHE-NE18) at either 14 or 21 days post-hatch had significantly lower levels of disease compared to birds from adjuvant only vaccinated hens. In addition, birds from hens immunized with rNetB(S254L) alone were significantly protected when challenged at 14 days post-hatch. These results demonstrate that maternal immunization with a NetB-enhanced toxoid vaccine is a promising method for the control of necrotic enteritis in young broiler chickens.     

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.     

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.     

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.     

Naturally acquired antibodies against Clostridium perfringens epsilon toxin in goats

Clostridium perfringens type D-producing epsilon toxin is a common cause of death in sheep and goats worldwide. Although anti-epsilon toxin serum antibodies have been detected in healthy non-vaccinated sheep, the information regarding naturally acquired antibodies in ruminants is scanty. The objective of the present report was to characterize the development of naturally acquired antibodies against C. perfringens epsilon toxin in goats. The levels of anti-epsilon toxin antibodies in blood serum of goat kids from two different herds were examined continuously for 14 months. Goats were not vaccinated against any clostridial disease and received heterologous colostrums from cows that were not vaccinated against any clostridial disease. During the survey one of these flocks suffered an unexpectedly severe C. perfringens type D enterotoxemia outbreak. The results showed that natural acquired antibodies against C. perfringens epsilon toxin can appear as early as 6 weeks in young goats and increase with the age without evidence of clinical disease. The enterotoxemia outbreak was coincident with a significant increase in the level of anti-epsilon toxin antibodies.     

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.     

A field study of naturally occurring specific antibodies against Clostridium perfringens alpha toxin in Norwegian broiler flocks

Necrotic enteritis (NE), a disease associated with high numbers of the intestinal bacterium Clostridium perfringens, is common in intensive broiler production. Antimicrobial feed additives may control the disease, but their use is now being questioned in many countries. A field study was undertaken at the end of 1997 to study the level of naturally occurring specific humoral immunity against phospholipase C (PLC; C perfringens alpha toxin) in Norwegian broiler flocks. Blood samples were collected at hatch from 61 study flocks, and the sampling was repeated for 56 of the same flocks at processing. The level of specific antibodies against PLC was analyzed in an enzyme-linked immunosorbent assay (ELISA) test. Data on production performance and weekly mortality were recorded. The relationship between the age of the hens and the level of specific maternal antibodies in the progenies was studied. The association between the level of the maternal antibodies and the production performance, including mortality, was analyzed. The level of specific antibodies against PLC in day-old broiler flocks was relatively high and varied considerably compared with the levels in the broilers at processing. The progenies from the oldest hens had significantly higher levels of specific antibodies than the chicks from younger hens. No outbreak of NE occurred during the study period, making it impossible to analyze the association between naturally occurring specific immunity against PLC and the occurrence of the disease. However, the results showed that the flocks with high titers of specific maternal antibodies against PLC had lower mortality during the production period than flocks with low titers.     

The role of penicillin G potassium in managing Clostridium perfringens in broiler chickens

The efficacy of penicillin G potassium (Pot-Pen) administered via drinking water to manage necrotic enteritis (NE) was investigated in a Clostridium perfringens (CP) challenge study using 1600 broiler chickens assigned to one of four treatment groups: nonchallenged, nonmedicated; challenged, nonmedicated; challenged, Pot-Pen 0.2 g/L; challenged, Pot-Pen 0.4 g/L. Overall mortality due to NE was significantly reduced among Pot-Pen-treated pens; mortality due to other causes did not differ among the treatment groups. Among all birds, growth performance parameters were significantly improved among Pot-Pen-treated pens. When considering birds randomly sacrificed 4 days post-Pot-Pen initiation, mean NE lesion scores were greatest among the challenged, nonmedicated pens; only one of 80 randomly sacrificed birds treated with Pot-Pen had NE lesions. Among the nonmedicated control pens, body weight (BW) was significantly greater among birds that did not have NE-associated lesions. When sacrificed birds were stratified by NE lesion score, there were no significant differences in BW among the treatment groups. Results of this study suggest that CP-associated subclinical disease can significantly reduce broiler performance. Furthermore, the positive effects of treatment with Pot-Pen appeared to be associated with the prevention and/or treatment of NE-specific lesions.     

Reproducible infection model for Clostridium perfringens in broiler chickens

Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments were conducted, each involving four groups of chickens with each group kept in separate isolators. A coccidial vaccine given at 10 times the prescribed dosage was used to promote the development of necrotic enteritis. In the first experiment, cultures of C. perfringens were mixed with the feed at day 9, 10, 11, and 12, and the coccidial vaccine was given at day 10, whereas in the second experiment, C. perfringens cultures were mixed with the feed at day 17, 18, 19, and 20, and the coccidial vaccine was given at day 18. Chickens were examined at day 9, 11, 12, and 15 (Experiment 1), and at day 17, 18, 20, and 24 (Experiment 2). There was no mortality in any of the groups; however, chickens in the groups receiving both coccidial vaccine and C. peifringens developed the subclinical form of necrotic enteritis, demonstrated by focal necroses in the small intestine, whereas chickens in control groups or groups receiving only coccidial vaccine or only C. perfringens cultures developed no necroses. The results underline the importance of predisposing factors in the development of necrotic enteritis.     

Perturbations of the ileal mycobiota by necrotic enteritis in broiler chickensOA

Background: Intestinal microbiota is critical for maintaining animal health and homeostasis. However, involvement of the fungal community, also known as the mycobiota, in animal health and disease is poorly understood. This study was aimed to examine the association between the intestinal mycobiota and the severity of necrotic enteritis(NE), an economically significant poultry disease.Methods: A total of 90 day-of-hatch Cobb broilers were infected with Eimeria maxima on d 10, followed by an oral...     

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.     

Incidence of Clostridium perfringens in broiler chickens and their environment during production and processing.

During a calendar year, a study was conducted involving 16 broiler flocks on four different farms, two farms belonging to each of two major U.S. poultry integrators. As determined by the detection of Clostridium perfringens in fecal or cecal samples, 15 (94%) of the flocks became positive for this bacterial enteropathogen, and only one remained negative throughout the 6-to-8-wk rearing period. Paper pads beneath chicks that were transported from the hatchery to the rearing house were contaminated with C. perfringens in 15 (94%) of the flocks. When sampled biweekly through grow out, 13 of the flocks were C. perfringens positive at 2 wk of age. These results suggest that colonization of the intestinal tract of broilers by C. perfringens is an early event. Of the environmental samples, all but feed in the hopper were contaminated before placement for at least one of the rearing periods. All sample types were contaminated at some point during the 6-to-8-wk grow-out period. Of the on-farm environmental samples, the highest incidences (percentage positive) of C. perfringens were detected in wall swabs (53%), fan swabs (46%), fly strips (43%), dirt outside the house entrance (43%), and swabs of workers' boots (29%). Birds were usually transported to the processing plant in coops that were already contaminated with C. perfringens. In the plant, C. perfringens was isolated more frequently from samples of scald water than from those of chill water. Clostridium perfringens was recovered from broiler carcasses after chilling in 13 (81%) of the 16 flocks. The proportion of C. perfringens-positive carcasses for the contaminated flocks ranged from 8% to 68% with a mean of 30%.     

Influence of Clostridium perfringens and its toxin in germ-free chickens

Twenty-one of 56 germ-free chickens died after receiving an oral inoculation of a broth culture of Clostridium perfringens. At necropsy there was detachment and disruption of the epithelial layer at the tips of villi and sloughed epithelial cells in the duodenum. These are characteristic lesions of necrotic enteritis. Germ-free chickens receiving either purified alpha-toxin or the supernatant of broth cultures of C perfringens died, but no bird died after receiving supernatant of broth cultures neutralised by anti-alpha-toxin serum. It was concluded that alpha-toxin of C perfringens was the cause of death in young germ-free chickens.     

Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks.

Two trials were conducted to study the effects of a competitive exclusion (CE) product BROILACT and the anticoccidial narasin on the incidence of necrotic enteritis (NE), the numbers of Clostridium perfringens (CP) in the caeca of broiler chicks and the performance of the birds. In trial 1 the effects of type of protein and partial replacement of a narasin containing diet with whole wheat were also studied. All groups of chicks were studied up to the point of slaughter at 43 days of age and after evisceration in a processing plant to determine slaughter yield. In trial 1, statistically significant results included the following: CE-treatment reduced total mortality, and incidence of NE, on diet containing animal but not vegetable protein. Caecal carriage of CP was also reduced, while slaughter yield increased. Narasin reduced caecal carriage of CP and increased both growth rate and slaughter yield in both trials. Whole wheat replacement improved feed conversion but reduced bird growth rate. In trial 2, both CE-treatment and narasin influenced feed intake, CE-treatment significantly only at days 22 and 44. Narasin improved feed conversion until 5 weeks of age and CE-treatment did so until 22 days of age. In both trials, there was also an interaction effect indicating that CE-treatment increased slaughter yield for birds that were not fed narasin.